DNA and RNA Polymerases with
Expanded Substrate Scope:
Synthesis of Modified Nucleic Acids
Using Engineered Polymerases Generated
by Directed Evolution
Dissertation
zur Erlangung des akademischen Grades
eines Doktors der Naturwissenschaften
(Dr. rer. nat.)
vorgelegt von
Vanessa Siegmund
an der Universität Konstanz
Mathematisch-Naturwissenschaftliche Sektion
Fachbereich Chemie
Konstanz, 2013
Konstanzer Online-Publikations-System (KOPS)
URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-248424
Tag der mündlichen Prüfung: 24. Mai 2013
1. Referent: Prof. Dr. Andreas Marx
2. Referent: Prof. Dr. Jörg Hartig
II
Parts of this work have been published in:
Chem. Commun. 2012, 48(79), 98709872.
V. Siegmund, T. Santner, R. Micura, A. Marx
“Screening mutant libraries of T7 RNA polymerase
for candidates with increased acceptance of 2’modified nucleotides.”
Chem. Eur. J. 2012, 18(3), 869-879.
M. Maiti, V. Siegmund, M. Abramov,
E. Lescrinier, H. Rosemeyer, M. Froeyen,
A. Ramaswamy, A. Ceulemans, A. Marx,
P. Herdewijn “Solution structure and conformational
dynamics of deoxyxylonucleic acids (dXNA): an
orthogonal nucleic acid candidate.”
Chem. Sci. 2011, 2(11), 2224-2231.
V. Siegmund, T. Santner, R. Micura, A. Marx
“Enzymatic synthesis of 2’-methylseleno-modified
RNA.”
Other publications:
Bioorg. Med. Chem. 2012, 20(7), 2416-2418.
T. Santner, V. Siegmund, A. Marx, R. Micura “The
synthesis of 2’-methylseleno adenosine and guanosine
5’-triphosphates."
Bioorg. Med. Chem. Lett. 2012, 22(9), 31363139.
B. Holzberger, J. Strohmeier, V. Siegmund, U.
Diederichsen, A. Marx “Enzymatic synthesis of 8vinyl- and 8-styryl-2’-deoxyguanosine modified DNA
– novel fluorescent molecular probes."
III
IV
Danksagung
Die vorliegende Doktorarbeit wurde im Zeitraum von Juni 2009 bis Januar 2013 in der Arbeitsgruppe
von Prof. Dr. Andreas Marx am Fachbereich Chemie der Universität Konstanz angefertigt. Die
Unterstützung von verschiedenen Leuten und die Zusammenarbeit mit meinen Kollegen hat zum
erfolgreichen Abschluss dieser Arbeit beigetragen. Dafür möchte ich mich an dieser Stelle bedanken.
Mein größter Dank geht an meinem Doktorvater Prof. Dr. Andreas Marx. Er hat mich in seine
Arbeitsgruppe aufgenommen und mich die ganzen Jahre über großartig unterstützt und motiviert. Das
von ihm gestellte Thema meiner Arbeit hat mich von Anfang an begeistert und mich immer wieder
aufs Neue gefordert. Ich möchte mich ebenfalls bei unseren Kooperationspartnern von der Universität
Innsbruck Prof. Dr. Ronald Micura und Tobias Santner für die gute und erfolgreiche Zusammenarbeit
bedanken.
Ich möchte mich bei Prof. Dr. Jörg Hartig für die Übernahme des Zweitgutachtens sowie bei Prof. Dr.
Martin Scheffner für die Übernahme des Prüfungsvorsitzes bedanken.
Der gesamten Arbeitsgruppe Marx danke ich für die herzliche Aufnahme in die Gruppe, den
Zusammenhalt, die Unterstützung und die tollen AG-Ausflüge und -Aktivitäten. Es hat immer viel
Spaß mit Euch gemacht und ich habe mich sehr wohl in Konstanz gefühlt! Ebenfalls danke ich Euch
für den offenen, wissenschaftlichen Austausch. Besonders möchte ich mich bei Samra, Anna, Sascha,
Bac, Nina, Nadine, Matze, Anna-Lena, Tobias, Norman, Holger, Hacker, Karin und Konrad bedanken.
Es war so schön mit Euch und ich werde noch lange an die tolle Zeit zurückdenken.
Meinen Laborkollegen Bac, Nadine, Sabrina, Nina und Daniel möchte ich ganz herzlich für die
angenehme, lockere Arbeitsatmosphäre und für ihre Unterstützung danken. Des Weiteren möchte ich
Janina Hendrick und Sandra Hess, meinen Mitarbeiterpraktikanten, für ihre Mitarbeit an
verschiedenen Teilprojekten dieser Arbeit danken.
Ich danke allen Freunden, Kollegen und lieben Menschen, mit denen ich gerne meine Freizeit
verbracht habe, für Eure Unterstützung und für die tolle Zeit am Bodensee!
Meiner Mutter Monika danke ich für Ihre Unterstützung während des Studiums und während der
Doktorarbeit.
V
VI
Summary
Modified RNA is used in a series of nucleic acid-based, cutting-edge technologies such as
antisense oligonucleotide, small interfering RNA (siRNA) or as aptamer. Furthermore, modifications
within RNA are used for studying RNA structure and dynamics. The 2’-position of the sugar moiety is
a desirable position for introducing modifications within the RNA target because modifications
attached to this position provide the RNA with desired properties such as increased stability.
Additionally, the 2’-position is important because it can be advantageous for introducing selenium into
RNA for use in X-ray crystallographic structure determination. 2’-Methylseleno (2’-SeCH3)-modified
RNA is accessible by traditional chemical synthesis, and thus limited in its available length due to the
inherent limitations of the synthesis on solid support. In the present work, the enzymatic synthesis of
2’-SeCH3-modified RNA has been elaborated by using engineered variants of T7 RNA polymerase.
This approach provides access to long selenium modified RNA, which cannot be accomplished by
chemical synthesis. Furthermore, this research shows the effectiveness of an established fluorescence
readout-based screening assay that was used to identify a variant of T7 RNA polymerase with an
increased acceptance of 2’-SeCH3-NTPs by directed evolution.
First, the enzymatic synthesis of 2’-SeCH3-modified RNA was investigated by using two T7
RNA polymerase mutants known to have a high tolerance for 2’-modified NTPs. These investigations
were conducted in collaboration with Prof. Dr. Ronald Micura from Innsbruck University in Austria,
where his research team assisted in synthesizing the four 2’-SeCH3-NTPs. In vitro transcription
reactions were performed in the presence of 2’-SeCH3-UTP or 2’-SeCH3-CTP involving the T7 RNA
polymerase mutants previously reported by Sousa (Y639F, H784A)1 and Ellington (E593G, Y639V,
V685A, H784G)2. It was discovered that both mutants can incorporate the 2’-SeCH3-modified
nucleotides into an 89-mer transcript at various positions, whereas the wildtype T7 RNA polymerase
fails to do so. Both mutants possess amino acid substitutions at position Y639 and H784, two residues
known to be involved in nucleotide discrimination.3,
4
Encouraged by these results, saturation
mutagenesis of amino acid positions Y639 and H784 was applied to construct a library comprising of
3200 variants. This library was screened for active T7 RNA polymerase variants by using a screening
assay based on fluorescence readout after transcription by using the RNA specific stain SYBR Green
II. The assay was established in 384-well plates to screen mutant libraries of T7 RNA polymerase in
high-throughput directly with diluted E. coli lysates containing the overexpressed variants. The
screening procedure was validated by screening the first library and then used to screen a second
mutant library composed of 1600 variants. The second mutant library was constructed by random
mutagenesis based on the Ellington mutant (E593G, Y639V, V685A, H784G) using error-prone PCR.
Active variants from both libraries were purified and tested in
32
P-based transcription assays for
increased incorporation efficiencies of the 2’-SeCH3-modified nucleotides. In doing so, a T7 RNA
polymerase variant from the second library was identified, referred to as 2P16, that exhibits an
VII
increased acceptance for the tested 2’-SeCH3-NTPs compared to the parental mutant from which it
derived. DNA sequencing revealed that this 2P16 has seven mutations in addition to the four initial
ones, totalling 11 mutations that surprisingly did not affect its ability to polymerize natural NTPs.
These results highlight the benefit of directed protein evolution and show that the established
screening system is well-suited to identify T7 RNA polymerase variants with altered properties like
the ability to incorporate 2’-modified nucleotides. Furthermore, this approach resulted in the
identification of a T7 RNA polymerase mutant that can further be used for the efficient enzymatic
synthesis of 2’-SeCH3-modified RNA for application in X-ray crystallography.
The second part of this work focuses on the enzymatic synthesis of four unnatural nucleic acid
polymers comprising of CeNA, HNA, ANA and dXNA (cyclohexenyl nucleic acid, 1,5anhydrohexitol nucleic acid, arabino nucleic acid and deoxyxylo nucleic acid). The investigation used
a selection of DNA polymerases and mutants of eukaryotic, prokaryotic, and archaic origin
representing different polymerase families. Over the past two decades, the scientific community has
intensively studied the chemical and enzymatic synthesis, the reverse transcription and structural
characteristics of synthetic genetic polymers, also referred to as xeno-nucleic acids (XNA). This
research was originally driven by the question of how life evolved on earth and why DNA and RNA
were naturally selected over other possible information storage systems. In this context, it was
investigated in the present work whether DNA polymerase mutants with broadened substrate spectra,
which had either been generated by site-directed mutagenesis or by directed protein evolution, are able
to synthesize the unnatural nucleic acids in a DNA-dependent manner. Homopolymer formation of the
four different sugar-modified XNAs was examined in
32
P-based primer extension assays in order to
identify DNA polymerases that show potential to polymerize the respective modified nucleotides. In
addition to testing 2’-deoxyxyloadenosine 5’-triphosphate (dXATP), four sugar modified thymidine
5’-triphosphates were also tested: hTTP, araTTP, ceTTP and dXTTP. The investigations revealed that
family-B polymerases and mutants were able to efficiently incorporate arabino, cyclohexenyl and
hexitol nucleotides into a growing XNA strand, whereas family-A polymerases had significant
problems with the sugar-modified nucleotides. Furthermore, it was observed that the tested DNA
polymerase mutants showed increased incorporation behaviours of the nucleotide analogues compared
to their respective wildtype polymerase. These results are promising and indicate that family-B
polymerases might be well-suited to evolve into DNA-dependent XNA polymerases. The
investigations on the DNA-dependent enzymatic synthesis of deoxyxylose nucleic acids (dXNA) have
revealed that dXATP and dXTTP are poor substrates for all of the tested DNA polymerases
irrespective of their original polymerase family. These observations are consistent with the proposed
orthogonality of dXNA. Nevertheless, a mutant of human DNA polymerase beta was observed to
efficiently incorporate and elongate the tested deoxyxylose nucleotides and might therefore be an
interesting candidate to develop into a DNA-dependent dXNA polymerase using directed protein
evolution.
VIII
Zusammenfassung
Modifizierte RNA findet Anwendung in einer Reihe von Nukleinsäure-basierten
Spitzentechnologien wie als Antisense Oligonukleotid, small interfering RNA (siRNA) und als
Aptamer. Außerdem werden RNA Modifikationen bei Methoden zur Strukturaufklärung und zur
Beobachtung der RNA-Dynamik verwendet. Die 2’-Position der Zuckereinheit ist eine beliebte
Position um Modifikationen in eine RNA Zielsequenz einzubringen, da Modifikationen an dieser
Position der RNA gewünschte Eigenschaften wie eine erhöhte Stabilität verleihen. Zusätzlich hat sich
die 2’-Position als vorteilhaft für das Einbringen von Selen in RNA zur Anwendung in der
Röntgenstrukturanalyse erwiesen. 2’-Selenomethyl (2’-SeCH3)-modifizierte RNA ist bislang nur über
chemische Synthese zugänglich und aus diesem Grund in der verfügbaren Länge begrenzt, da die
Festphasensynthese diese methodenbedingte Limitierung mit sich bringt. In der vorliegenden Arbeit
wurde die enzymatische Synthese von 2’-SeCH3-modifizierter RNA unter Verwendung von
veränderten Varianten der T7 RNA-Polymerase erarbeitet. Diese Annäherung eröffnet den Zugang zu
längeren Selen-modifizierten RNA Molekülen, die nicht über chemische Synthese hergestellt werden
können. Darüber hinaus konnte in dieser Arbeit die Wirksamkeit eines Fluoreszenz-basierten
Durchmusterungsassays gezeigt werden, der dazu benutzt wurde eine T7 RNA-Polymerase Variante
mit erhöhter Akzeptanz für 2’-SeCH3-NTPs über gerichtete Evolution zu identifizieren.
Zunächst wurde die enzymatische Synthese von 2’-SeCH3-modifizierter RNA unter
Verwendung von zwei T7 RNA-Polymerase Mutanten untersucht, die bekannt sind eine erhöhte
Toleranz gegenüber 2’-modifizierten NTPs aufzuweisen. Diese Untersuchungen wurden in
Zusammenarbeit mit Prof. Dr. Ronald Micura von der Universität Innsbruck in Österreich
durchgeführt, in dessen Arbeitsgruppe die Synthese der vier 2’-SeCH3-NTPs erarbeitet wurde. Es
wurden in vitro Transkriptionsreaktionen in der Anwesenheit von 2’-SeCH3-UTP oder 2’-SeCH3-CTP
mit T7 RNA-Polymerase Mutanten durchgeführt, die zuvor von Sousa (Y639F, H784A)1 und
Ellington (Y639V, H784G, E593G, V685A)2 beschrieben wurden. Es wurde festgestellt, dass beide
Mutanten in der Lage waren die 2’-SeCH3-modifizierten Nukleotide in ein 89 Nukleotide langes
Transkript an mehreren verschiedenen Positionen einzubauen, wohingegen die Wildtyp T7 RNAPolymerase nicht dazu in der Lage war. Ermutigt von diesen Ergebnissen wurde daraufhin eine
Mutantenbibliothek durch Sättigungsmutagenese an den beiden Aminosäurepositionen Y639 und
H784 erstellt, die 3200 Varianten umfasste. Die Bibliothek wurde nach aktiven T7 RNA-Polymerase
Varianten durchmustert indem ein Durchmusterungsassay verwendet wurde, der auf dem Auslesen
von Fluoreszenz mit Hilfe des RNA-spezifischen Farbstoffs SYBR Green II nach der Transkription
basiert. Dieser Durchmusterungsassay wurde in 384-Lochplatten entwickelt, um Mutantenbibliotheken
der T7 RNA-Polymerase im Hochdurchsatz unter direkter Verwendung von verdünnten E. coli
Lysaten zu durchmustern, die die überexprimierten Varianten enthielten. Der Durchmusterungsprozess
wurde bei der Durchmusterung der ersten Bibliothek validiert und im Anschluss dazu genutzt eine
IX
zweite Mutantenbibliothek zu durchmustern, die aus 1600 Varianten bestand. Diese zweite
Mutantenbibliothek
wurde
über
randomisierte
Mutagenese
ausgehen
von
der
Ellington
Vierfachmutante über fehleranfällige PCR (error-prone PCR) hergestellt. Aktive Varianten aus beiden
Bibliotheken
wurden
aufgereinigt
und
in
32
P-basierten
Transkriptionsassays
auf
erhöhte
Einbaueffizienzen von 2’-SeCH3-modifizierten Nukleotiden untersucht. Auf diese Weise konnte eine
T7 RNA-Polymerase Variante aus der zweiten Bibliothek identifiziert werden, die eine erhöhte
Akzeptanz der untersuchten 2’-SeCH3-NTPs im Vergleich zur Ausgangsmutante aufweist und die im
Folgenden als 2P16 bezeichnet wurde. Die Sequenzierung des Gens dieser Mutante brachte zum
Vorschein, dass diese zusätzlich noch sieben weitere Mutationen zu den vier anfänglichen Mutationen
aufweist und diese 11 Mutationen erstaunlicherweise nicht ihre Fähigkeit beeinflussen natürliche
NTPs zu polymerisieren. Diese Ergebnisse demonstrieren ein weiteres Mal die Leistungsfähigkeit von
gerichteter Protein Evolution Enzyme mit gewünschten Eigenschaften zu generieren. Außerdem
konnte gezeigt werden, dass das etablierte Durchmusterungssystem gut dafür geeignet ist um T7
RNA-Polymerase Varianten mit veränderten Eigenschaften, wie die Fähigkeit zum Einbau von 2’modifizierten Nukleotiden, zu identifizieren. Hinzu kommt, dass dieses Vorgehen zur Identifizierung
einer T7 RNA-Polymerase Variante führte, die für die effiziente enzymatische Synthese von 2’SeCH3-modifizierter RNA für die Anwendung in der Röntgenstrukturanalyse verwendet werden kann.
Im zweiten Teil dieser Arbeit wurde die enzymatische Synthese von vier unnatürlichen
Nukleinsäurepolymeren, einschließlich CeNA, HNA, ANA und dXNA (cyclohexenyl nucleic acid,
1,5-anhydrohexitol nucleic acid, arabino nucleic acid und deoxyxylo nucleic acid) untersucht. Die
Untersuchungen wurden unter Verwendung von einer Auswahl an DNA-Polymerasen und Mutanten
eukaryotischen, prokaryotischen und archaeischen Ursprungs aus verschiedenen Polymerase Familien
durchgeführt. Im Laufe der letzten Jahrzehnte wurde die chemische und enzymatische Synthese, die
reverse Transkription und die strukturellen Eigenschaften von synthetischen genetischen Polymeren,
die auch als Xeno-Nukleinsäuren (XNAs) bezeichnet werden, intensiv von der Wissenschaft
untersucht. Die Studien sollen dazu beitragen das allgemeine Wissen bezüglich der Entstehung von
Leben auf der Erde und der natürlichen Auswahl von DNA und RNA gegenüber anderen
Informationssystemen zur Speicherung der Erbinformation zu erweitern. In diesem Zusammenhang
wurde in der vorliegenden Arbeit untersucht, ob DNA-Polymerase Mutanten mit erweitertem
Substratspektrum, die entweder über ortsgerichtete Mutagenese oder über gerichtete Protein Evolution
hergestellt wurden, in der Lage sind die unnatürlichen Nukleinsäuren in einer DNA-abhängigen Art
und Weise zu synthetisieren. Es wurde dabei die Homopolymerbildung der vier verschiedenen
zuckermodifizierten XNAs in
32
P-basierenden Primerverlängerungsreaktionen untersucht, um nach
Möglichkeit DNA-Polymerasen zu identifizieren, die ein Potenzial zur Polymerisierung der
modifizierten Nukleotide aufweisen. Zusätzlich zu 2’-Deoxyxyloadenosin 5’-Triphosphat (dXATP)
wurden ebenfalls vier zuckermodifizierte Thymidin Triphosphate getestet: hTTP, araTTP, ceTTP und
dXTTP. Die Untersuchungen ergaben, dass B-Familie Polymerasen und Mutanten in der Lage waren
X
arabino-, cyclohexenyl- und hexitol-modifizierte Nukleotide effizient in einen wachsenden XNA
Strang einzubauen, wohingegen Polymerasen der A-Familie erhebliche Probleme mit den
zuckermodifizierten Nukleotiden hatten. Es konnte außerdem beobachtet werden, dass die
untersuchten DNA-Polymerase Mutanten ein besseres Einbauverhalten der Nukleotid-Analoga
aufwiesen als die jeweils zugehörige Wildtyp-Polymerase. Diese Ergebnisse sind vielversprechend
und zeigen, dass B-Familie Polymerasen gut dafür geeignet wären um aus ihnen DNA-abhängige
XNA-Polymerasen zu erzeugen. Die Untersuchungen bezüglich der DNA-abhängigen enzymatischen
Synthese von Deoxyxylose Nukleinsäuren (dXNA) haben gezeigt, dass dXATP and dXTTP schlechte
Substrate für alle getesteten DNA-Polymerasen sind, unabhängig davon welcher Polymerasen Familie
sie angehören. Diese Beobachtungen sind übereinstimmend mit der vermuteten Orthogonalität von
dXNA. Es konnte dennoch beobachtet werden, dass eine Mutante der humanen DNA-Polymerase beta
die getesteten Deoxyxylose-Nukleotide effizient einbauen und verlängern konnte und daher ein
interessantes Ausgangsenzym darstellt, um über gerichtete Protein Evolution eine DNA-abhängige
dXNA-Polymerase zu entwickeln.
XI
XII
Table of contents
1
Introduction........................................................................................................1
1.1
Structure of DNA, RNA and other synthetic genetic polymers.................................1
1.1.1
DNA ................................................................................................................................1
1.1.2
RNA.................................................................................................................................2
1.1.3
Synthetic genetic polymers..............................................................................................3
1.2
1.2.1
1.3
DNA polymerases ..........................................................................................................4
DNA polymerase mechanism..........................................................................................5
RNA polymerases ..........................................................................................................6
1.3.1
Transcription....................................................................................................................7
1.3.2
T7 RNA polymerase........................................................................................................8
1.4
Directed evolution........................................................................................................10
1.4.1
General ..........................................................................................................................10
1.4.2
Directed evolution of nucleic acid polymerases............................................................11
1.5
Modified RNA and its applications............................................................................12
1.5.1
Overview .......................................................................................................................12
1.5.2
Synthesis of modified RNA...........................................................................................14
2
Aim of this Work..............................................................................................15
3
Results and Discussion.....................................................................................17
3.1
Enzymatic Synthesis of 2’-Selenium-modified RNA Using Engineered
RNA Polymerases ........................................................................................................17
3.1.1
Introduction ...................................................................................................................17
3.1.2
Construction of T7 RNA polymerase wildtype recombinant plasmid for
protein expression in E. coli ..........................................................................................20
3.1.3
Generation of T7 RNA polymerase mutants M1 (Y639F, H784A)
and M2 (E593G, Y639V, V685A, H784G)...................................................................20
3.1.4
Heterologous expression and purification of T7 RNA polymerases
in E. coli BL21-Gold (DE3) ..........................................................................................20
3.1.5
In vitro transcription of relevant RNA sequences using natural NTPs
and 2’-SeCH3-UTP ........................................................................................................21
3.1.6
Synthesis of 2’-SeCH3-modifed RNA by T7 RNA polymerase mutants M1
and M2...........................................................................................................................25
3.1.7
Development of a fluorescence readout based high-throughput screening
assay for the identification of active T7 RNA polymerase variants from
mutant libraries..............................................................................................................29
XIII
3.1.8
Construction of a T7 RNA polymerase mutant library by random mutagenesis
of amino acid positions Y639 and H784....................................................................... 32
3.1.9
Expression and screening of the mutant library in which amino acid positions
Y639 and H784 were randomized for active variants .................................................. 33
3.1.10
Purification and characterization of identified hits regarding the incorporation
of 2’-modified NTPs..................................................................................................... 35
3.1.11
Construction of a randomized T7 RNA polymerase mutant library based
on mutant M2 (E593G, Y639V, V685A, H784G) by error-prone PCR ....................... 38
3.1.12
Expression and screening of the randomized mutant library for active
variants.......................................................................................................................... 39
3.1.13
Purification and characterization of identified hits regarding the incorporation
of 2’-modified NTPs..................................................................................................... 41
3.1.14
3.2
Discussion of the results and conclusion ...................................................................... 45
Studies on the Enzymatic Synthesis of Synthetic Genetic Polymers ...................... 51
3.2.1
Introduction................................................................................................................... 51
3.2.2
Expression and purification of KOD exo- DNA polymerase, mutant L408Q
and mutant L408Q, A485L........................................................................................... 52
3.2.3
Incorporation studies of araTTP, hTTP, xyloTTP and ceTTP by various
DNA polymerases and mutants .................................................................................... 53
3.2.4
Incorporation studies of 2’-deoxyxylo-ATP and xyloTTP by various
DNA polymerases and mutants .................................................................................... 55
3.2.5
4
Discussion of the results and conclusion ...................................................................... 58
Materials and Methods ................................................................................... 61
4.1
XIV
Materials...................................................................................................................... 61
4.1.1
General.......................................................................................................................... 61
4.1.2
Chemicals used for molecular biology ......................................................................... 61
4.1.3
Reagents used for chemical synthesis........................................................................... 62
4.1.4
Nucleotides and radiochemicals ................................................................................... 63
4.1.5
Oligonucleotides ........................................................................................................... 63
4.1.6
DNA, RNA and Protein Standards ............................................................................... 63
4.1.7
Enzymes and Proteins................................................................................................... 64
4.1.8
Kits................................................................................................................................ 64
4.1.9
Bacterial strains and plasmids....................................................................................... 65
4.1.10
Electrophoresis buffers and solutions ........................................................................... 65
4.1.11
Buffers used in enzymatic reactions ............................................................................. 68
4.1.12
Buffers and solutions used for screening ...................................................................... 69
4.1.13
Buffers and solutions used for protein purification ...................................................... 69
4.1.14
Media.............................................................................................................................71
4.1.15
Antibiotics .....................................................................................................................71
4.1.16
Instruments ....................................................................................................................72
4.1.17
Disposables....................................................................................................................73
4.2
Methods ........................................................................................................................74
4.2.1
Chemical synthesis ........................................................................................................74
4.2.1.1
General......................................................................................................................74
4.2.1.2
Chemical synthesis of 2’-O-methyl-2’-deoxyuridine-5’-triphosphate .....................75
4.2.2
Molecular Cloning.........................................................................................................75
4.2.2.1
Polymerase chain reaction (PCR) .............................................................................75
4.2.2.2
Site-directed mutagenesis .........................................................................................76
4.2.2.3
Multi site-directed mutagenesis ................................................................................76
4.2.2.4
PCR with randomized primers..................................................................................76
4.2.2.5
Overlap extension PCR.............................................................................................77
4.2.2.6
Error-prone PCR .......................................................................................................77
4.2.2.7
Restriction digest of double-stranded DNA..............................................................77
4.2.2.8
Dephosphorylation of double-stranded DNA ...........................................................78
4.2.2.9
DNA Ligation ...........................................................................................................78
4.2.2.10
Sequencing of double-stranded DNA .......................................................................78
4.2.2.11
Analytical agarose gel electrophoresis......................................................................78
4.2.2.12
Preparative agarose gel electrophoresis ....................................................................79
4.2.2.13
SDS-PAGE ...............................................................................................................79
4.2.2.14
Denaturing PAGE .....................................................................................................79
4.2.2.15
Preparative denaturing PAGE...................................................................................79
4.2.3
Oligonucleotide based methods.....................................................................................80
4.2.3.1
Dephosphorylation of ssRNA ladder ........................................................................80
4.2.3.2
5’-Phosphorylation of oligonucleotides using [γ-32P]ATP .......................................80
4.2.3.3
Ethanol precipitation.................................................................................................81
4.2.3.4
Phenol-chloroform extraction ...................................................................................81
4.2.3.5
Electrospray ionization MS (ESI-MS) of oligonucleotides ......................................81
4.2.3.6
Determination of DNA and RNA concentration ......................................................81
4.2.4
Microbiological methods...............................................................................................81
4.2.4.1
Preparation of electrocompetent E. coli cells ...........................................................81
4.2.4.2
Transformation of electrocompetent E. coli cells .....................................................82
4.2.4.3
LB agar plate cultures ...............................................................................................82
4.2.4.4
Liquid cultures ..........................................................................................................82
4.2.4.5
Plasmid DNA isolation from liquid cultures ............................................................82
XV
4.2.4.6
Colony PCR ............................................................................................................. 83
4.2.4.7
E. coli glycerol stock preparation............................................................................. 83
4.2.5
Biochemical methods.................................................................................................... 83
4.2.5.1
Expression of T7 RNA polymerases........................................................................ 83
4.2.5.2
Purification of T7 RNA polymerases....................................................................... 84
4.2.5.3
Expression of T7 RNA polymerase mutant libraries ............................................... 84
4.2.5.4
Preparation of E. coli BL21 lysates for screening of T7 RNA polymerase
mutant libraries ........................................................................................................ 84
4.2.5.5
Purification of T7 RNA polymerase hits in 96-well filter plates ............................. 84
4.2.5.6
Expression and purification of T7 RNA polymerase hits ........................................ 85
4.2.5.7
Expression of KOD DNA polymerases ................................................................... 85
4.2.5.8
Purification of KOD DNA polymerases .................................................................. 86
4.2.5.9
Determination of protein concentrations via Bradford protein assay....................... 86
4.2.6
Assays ........................................................................................................................... 86
4.2.6.1
In vitro-transcription assays ..................................................................................... 86
4.2.6.2
Time course experiment for the incorporation of 2’-SeCH3-NTPs.......................... 87
4.2.6.3
Quantification of 2’-SeCH3-modified RNA............................................................. 87
4.2.6.4
T7 RNA polymerase screening assay ...................................................................... 87
4.2.6.5
Primer extension reactions using araTTP, hTTP, xyloTTP and ceTTP................... 88
4.2.6.6
Primer extension reactions using xyloTTP and xylo-dATP..................................... 88
5
Appendix .......................................................................................................... 89
5.1
Sequences..................................................................................................................... 89
5.1.1
5.1.1.1
Primer used for site-directed-mutagenesis ............................................................... 89
5.1.1.2
Primer used for cloning and PCR............................................................................. 90
5.1.1.3
Oligonucleotides used for in vitro transcription....................................................... 90
5.1.1.4
Primer and templates used for primer extension reactions ...................................... 91
5.1.1.5
Primer used for DNA sequencing ............................................................................ 91
5.1.2
Plasmids........................................................................................................................ 92
5.1.2.1
pGDR11 ................................................................................................................... 92
5.1.2.2
pET24a (+) ............................................................................................................... 93
5.1.3
Expression plasmids ..................................................................................................... 94
5.1.3.1
T7 RNAP wildtype-pGDR11................................................................................... 94
5.1.3.2
KOD (exo-) wildtype-pET24a(+)............................................................................. 95
5.1.4
XVI
Oligonucleotide primer and templates .......................................................................... 89
Protein sequences.......................................................................................................... 95
5.1.4.1
T7 RNA polymerase wildtype ................................................................................. 95
5.1.4.2
T7 RNA polymerase mutant M1 (Y639F, H784A) ................................................. 96
5.1.4.3
T7 RNA polymerase mutant M2 (E593G, Y639V, V685A, H784G) ......................96
5.1.4.4
T7 RNA polymerase mutant 2P16 (I119V, G225S, K333N, D366N,
F400L, E593G, Y639V, S661G, V685A, H784, F880Y).........................................96
5.2
6
5.1.4.5
KOD exo- DNA polymerase wildtype......................................................................97
5.1.4.6
KOD exo- DNA polymerase mutant L408Q ............................................................97
5.1.4.7
KOD exo- DNA polymerase mutant L408Q, A485L...............................................97
Abbreviations...............................................................................................................98
References.......................................................................................................101
XVII
XVIII
1. Introduction
1 Introduction
1.1
Structure of DNA, RNA and other synthetic genetic polymers
1.1.1
DNA
DNA (desoxyribonucleic acid) is a biopolymer that is used in all living organisms to store and
replicate
genetic
information.
It
is
composed
of
four
different
building
blocks,
the
desoxyribonucleotides that consist of 2’-deoxyribose, phosphate and of one of the four nucleobases
adenine (A), guanine (G), cytosine (C) and thymine (T). The 2’-deoxyribose sugar moieties and the
phosphates form together the DNA backbone via phosphodiester bonds whereas the nucleobases that
are attached to the 2’-deoxyribose are arranged in a well defined order to code for the genetic
information.5 The four nucleobases form specific base pairs by hydrogen bond interactions in which
adenine pairs with thymine and guanine with cytosine (Watson-Crick base paring). Due to these
specific base pairs, DNA has some unique properties: it can hybridize to a complementary DNA strand
to form a double strand and thus can serve as a template.6 Acoording to Watson and Crick that had
postulated the structure of DNA in 1953, double-stranded DNA forms a right-handed, antiparallel
double helix that exhibits a major and a minor groove.6, 7 In this double helix, the nucleobases are
orientated to the center of the helix. Depending on nucleotide sequence and environment, DNA can
form different types of structures.8, 9 These types differ in the number of nucleotides per helical turn,
the conformation of the ribose sugar and the geometry of the minor and major groove. The righthanded double helix adopts preferred a B-form DNA, but A-form and Z-form DNA have also been
reported.9
Figure 1.1: Chemical structures of DNA nucleobases (left) and Watson-Crick base pairing (right). Dashed lines
indicate hydrogen bond interactions.
1
1. Introduction
1.1.2
RNA
RNA (ribonucleic acid) represents a biopolymer that is involved in the transfer of encoded
genetic information required for gene expression in form of messenger RNA (mRNA). Furthermore, it
contributes to protein biosynthesis in form of transfer RNA (tRNA) and is a component of the
ribosome as ribosomal RNA (rRNA). In addition to these major roles, a large number of non-coding
RNA molecules (ncRNA) including small interfering RNA (siRNA), micro RNA (miRNA),
riboswitches and ribozymes are known nowadays to fulfill different regulatory or catalytic functions.
RNA is composed of four different building blocks, the ribonucleotides that consist of ribose,
phosphate and one of the four nucleobases adenine (A), guanine (G), cytosine (C) and uracil (U). The
ribose sugar moieties and the phosphates form together the RNA backbone via phosphodiester bonds
whereas the nucleobases are attached to the ribose.5 Consequently, the composition of RNA differs
from that of DNA in the presence of ribose instead of 2’-deoxyribose as sugar moiety and the presence
of uracil instead of thymin. In order to transfer genetic information that is encoded into DNA, RNA is
synthesized by RNA polymerases using the non-coding DNA strand as template. In this DNA-RNA
hybrid that adopts an A-form conformation,10 adenine pairs with uracil and guanine with cytosine by
Watson-Crick base pairing. Unlike DNA, mRNA mostly appears single-stranded where it can serve as
a template for protein biosynthesis. The fact that single stranded RNA can adopt complex threedimensional structures explains the diversity of its function. Nevertheless, double-stranded RNA is
stable and forms a right-handed, antiparallel double helix (A-from).5
Figure 1.2: Chemical structures of RNA nucleobases (left) and Watson-Crick base pairing in a DNA-RNA
hybrid structure (right). Dashed lines indicate hydrogen bond interactions.
2
1. Introduction
1.1.3
Synthetic genetic polymers
The chemical and enzymatic synthesis of synthetic genetic polymers, also termed xeno-nucleic
acids (XNAs), their reverse transcription and general structural characteristics have been extensively
studied over the last two decades.11 Motivated by the question of how life evolved on earth and why
DNA and RNA were selected by evolution over other possible nucleic acid structures, the research is
nowadays driven by the idea of synthetic biology and systems chemistry to create orthogonal
information storage systems, the design of orthogonal chromosomes and the generation of artificial
biological systems and organisms.12, 13 XNA exhibits a variety of chemical and structural modifications
relative to its natural counterpart. Systematic investigations on the chemical diversification of nucleic
acids initiated by Eschenmoser and colleagues resulted in the synthesis of completely novel synthetic
genetic polymers.14 Among these analogues can be found the tetrose-based TNA (α-L-threofuranosyl
nucleic acid), LNA (locked nucleic acids; 2’-O,4’-C-methylen-β-D-ribonucleic acids), HNA (1,4anhydro hexitol nucleic acids), CeNA (cyclohexenyl nucleic acids), GNA (glycerol nucleic acids),
dXNA (xylo nucleic acids), ANA (arabino nucleic acids), and FANA (2’-fluoro-arabino nucleic
acids).11
Figure 1.3: Chemical structures of synthestic genetic polymers (XNAs). ANA: arabino nucleic acids; FANA: 2’fluoro-arabinonucleic acids; LNA: locked nucleic acids; TNA: α-L-threofuranosyl nucleic acids; CeNA:
cyclohexenyl nucleic acids; HNA: 1,5-anhydrohexitol nucleic acids; dXNA: xylose nucleic acids; GNA: glycol
nucleic acids.
3
1. Introduction
Some of the synthetic genetic polymers synthesized and investigated so far are capable of self-pairing
and some of them are also able to cross-hybridize with DNA and/or RNA (Figure 1.4).15-19 These
XNAs with modified sugar-moieties use the four letter code to store their genetic information and can
thus retain the ability to communicate with extant biology by base pairing with DNA.
Figure 1.4: Structures of natural duplexes and that of different synthetic genetic polymers (XNA). (a) Natural
duplexes; (b) XNA-DNA hybrid structures; (c) XNA-RNA hybrid structures; (d) XNA-XNA structures. The
figure is taken from: Pinheiro, V. B. and P. Holliger (2012). "The XNA world: progress towards replication and
evolution of synthetic genetic polymers." Curr Opin Chem Biol, 16(3-4), 245-252.
1.2
DNA polymerases
DNA polymerases catalyze the formation of DNA in all living organism which is synthesized
during DNA replication, repair and recombination.20 DNA polymerases perform DNA synthesis by the
addition of 2’-deoxynucleotides to the free 3’-hydroxyl group of the growing DNA strand and
phosphodiester bond formation. DNA synthesis is accomplished in 5’-3’ direction and nucleotides
incorporated according to Watson-Crick base pairing. For initiating DNA synthesis, the polymerase
requires an oligonucleotide, the so-called primer, that is hybridized with the single-stranded DNA
template.21 During replication in eukaryotes and prokaryotes, a short RNA sequence is used as primer
4
1. Introduction
for elongation. This RNA primer is synthesized by specialized RNA polymerases called primases.22
The substrates of DNA polymerases are the 2’-deoxyribonucleoside-5’-triphosphates of adenine
(dATP), guanine (dGTP), cytosine (dCTP) and thymine (TTP).
DNA polymerases are composed of different domains that have different functions. The
polymerase domain resembles an open right hand and is divided into palm, thumb and fingers
subdomains.23,
24
Furthermore, some DNA polymerases have additional domains like the 5’-3’
exonuclease domain that catalyzes the removal of 2’-deoxynucleotides from an existing DNA strand
in 5’-3’ direction. DNA polymerases are categorized depending on their function: replicative, repair or
translesion synthesis polymerase. Furthermore, they are divided into families according to their
sequence homologies.25-27 These polymerase families comprise family-A, -B, -C, -D, -E, -X, -Y and
reverse transcriptases (RT). DNA polymerases from all kingdoms of life are assigned to these families.
Besides their important role in nature, DNA polymerases are used in many molecular biological and
biotechnological applications.28,
32
29
Methods like PCR30,
31
and PCR- based methods like real-time
33
PCR or quantitative real-time PCR are commonly used in the laboratory everyday life, in molecular
diagnostics and DNA sequencing techniques. The availablity of thermostable DNA polymerases of
prokaryotic and archaeaic origin is a basic requirement for these methods.
1.2.1
DNA polymerase mechanism
The catalytic cycle of nucleotide incorporation can be divided into different steps.21 First, the
DNA polymerase binds to the primer-template DNA complex. Upon binding of the incoming dNTP
the polymerase undergoes a conformational change to form a closed conformation. This step is crucial
for nucleotide discrimination and catalysis since the correct positioning of the catalytic residues and
the reactive 3’-hydroxyl group of the primer towards the bound dNTP in the active site is necessary
for formation of the phosphodiester bond.34, 35 Incorrect dNTPs are not fitting properly into the active
site geometry, have a lower affinity to the complex and are thus not well tolerated. This so-called
‘induced fit mechanism’ results in a tighter binding of the correct nucleotide ans provides in addition
to the Watson-Crick base pairing further discrimination of the correct base pair.36 After phosphodiester
bond formation, the polymerase undergoes another conformational change to release pyrophosphate
and to enter another round of catalysis.
The molecular mechanism by which all DNA polymerases catalyze the phosphoryltranfer
reaction was first stated by Steitz et al. in 1998.23 This general two-metal-ions mechanism postulates
the involvement of two divalent metal ions (A and B) promoting chemical catalysis. Metal ion A
interacts with the 3’-OH group of the previously incorporated 2’-desoxynucleotide and is supposed to
lower the pKa value of this hydroxyl group and thus facilitates its nucleophilic attack on the αphosphate of the incoming dNTP.37 Both metal ions A and B act as Lewis acids and are proposed to
stabilize the pentacovalent transition state that occurs during the course of this phosphoryl transfer
5
1. Introduction
reaction.23, 38 Metal ion B coordinates and stabilizes the β- and γ-phosphates and facilitates the release
of pyrophosphate. Two highly conserved aspartates located in the polymerase’s active site coordinate
these two divalent metal ions.39-41
Due to their important role in the replication of genetic information, the specificity of
replicative DNA polymerases in selecting the correct nucleotides is fundamental to genome integrity.
Structural studies involving isosteric nucleotides lacking hydrogen bonding capacity showed that
nucleotide selectivity is not only provided by hydrogen fond formation with the correct base pair
according to Watson and Crick, but that also the base pair and active site geometry contributes to the
selectivity of DNA polymerases.42-45 In this context, the model of an ‘active site tightness’ was
proposed. This model describes the active site of a high-fidelity DNA polymerase as a tight binding
pocket which geometry is complementary to that of the canonical base pairs. According to this, noncanonical base pairs result in steric clashes.46-48 In contrast, the active site of a low fidelity DNA
polymerase has been proposed to be less tight and more flexible to allow the incorporation of noncanonical base pairs. The selection of the correct nucleotide by replicative DNA polymerases is a very
complex process in which hydrogen bond interactions between the polymerase and the minor groove
of the DNA, base stacking and sterical effects contribute as well as Watson-Crick base pairing to the
high accuracy of these enzymes.35, 49 In addition to these intrinsic quality control mechanisms, some
DNA polymerases exhibit also a 3’-5’ exonuclease function named proof reading that removes
incorporated mispaired nucleotides from the extended DNA strand.35
1.3
RNA polymerases
In all organisms, RNA polymerases are the key enzymes for RNA synthesis catalyzing the
formation of RNA in a DNA-dependent manner. RNA polymerases can be found in all three domains
of life, but the number and composition of these proteins is varying.5 In prokaryotes, a single type of
RNA polymerase is responsible for the synthesis of the entire RNA. Eukaryotic transcription is
accomplished by three distinct types of RNA polymerases (RNAP I-III).50-52
These three
polymerases differ in the number and type of subunits they are composed of and in the class of RNA
they synthesize. In general, RNA polymerases can be divided into two classes: multisubunit
comprising eukaryotic, bacterial and archaeal RNA polymerases and single-subunit, including RNA
polymerases of bacteriophages, mitochondria and chloroplasts. These both classes of polymerases
share no significant sequence homology and are structural different. However, they undergo the
different stages of transcription in an identical manner and share a number of functional
characteristics.53-55 The synthesis of RNA is conducted in 5’-3’ direction by the addition of
ribonucleotides to the nascent RNA strand catalyzed by the RNA polymerase. The substrates for the
RNA polymerase are the ribonucleoside 5’-triphosphates (NTPs) of adenosine (ATP), guanosine
(GTP), cytidine (CTP) and uridine (UTP). The mechanism by which all RNA polymerases catalyze
6
1. Introduction
the polymerization of ribonucleotides to the 3’-OH group of the previously incorporated nucleotide is
identical to that observed for other nucleic acid polymerases.56 This two-metal-ion mechanism
involves two divalent metal ions that are complexed by two highly conserved aspartates located in the
RNA polymerase’s active site facilitating chemical catalysis.23 Although the chemistry of
polymerization of DNA and RNA polymerases is likely to be conserved, RNA polymerases are able to
incorporate the first ribonucleotide without the need for a primer oligonucleotide resulting in de novo
RNA synthesis.57, 58
1.3.1
Transcription
Transcription as the DNA-dependent RNA synthesis is a complex process that takes place in
an identical manner in eukaryotes and prokaryotes. It can be divided into three different stages:
initiation, elongation and termination.55 To initiate RNA synthesis, the RNA polymerase must
recognize a specific DNA promoter sequence which is located upstream (5’-direction) of the starting
site of transcription and bind to it. In prokaryotes, the promoter region is recognized by a subunit of
the RNA polymerase called sigma factor.5 In eukaryotes, the promoter region (TATA-Box) is not
directly recognized by the RNA polymerase but through a complex of transcription factors that bind to
the DNA and mediate the binding of other transcription factors and of the RNA polymerase.59, 60 Once
the RNA polymerase is bound, other transcription factors are recruited to form the mature
transcription complex.61 Transcription begins with the unwinding and separation of the two DNA
strands to form a transcription bubble. RNA is synthesized starting at the transcription initiation site
using the non-coding strand of the DNA as template and NTPs as substrates. In the elongation phase,
the RNA polymerase proceeds down the DNA template, synthesizing a full-length RNA transcript.
During the elongation process, the length of the DNA-RNA hybrid is maintained and the transcription
bubble moves along with the RNA polymerase down the template.62 As soon as the RNA polymerase
has reached the end of the transcript, the transcription bubble collapses, the transcript is released from
the complex and the polymerase dissociates from the DNA.
In prokaryotes, this transcription termination is accomplished by intrinsic terminator
sequences or by the termination factor rho. Intrinsic terminator sequences are part of the RNA
transcript and composed of a GC-rich sequence that forms a stem-loop structure followed by a
polyuridine region. This secondary structure destabilizes the DNA-RNA hybrid and the elongation
complex at the 5’-end and thus promotes dissociation of the polymerase and release of the RNA
transcript.63 Termination by the transcriptional termination factor rho is initiated by a Rho recognition
site on the synthesized RNA strand which recruites the rho factor protein as soon as it has emerged
from the exit site of the RNA polymerase. The rho factor then advances along the RNA in 5’-3’
direction driven by ATP-hydrolysis and thus causes the dissociation of the RNA polymerase from the
DNA template.64 In eukaryotes, termination is much more complex due to the fact that RNA is
7
1. Introduction
extensively post-transcriptional processed. Termination factors are required that bind to the
termination signal to interrupt the RNA polymerase during transcription and to release the nascent
RNA strand from the transcription complex.
1.3.2
T7 RNA polymerase
T7 RNA polymerase (T7 RNAP) is an enzyme frequently used in molecular biology for the
DNA-dependent preparation of specific RNA in vitro and for gene expression in vivo.65, 66 It was first
identified 1970 from bacteriophage T7 infected E. coli cells.67, 68 T7 RNAP is a single subunit RNA
polymerase composed of 883 amino acids with a molecular weight of 99 kDa. T7 RNAP has a robust
activity, is stringent for its promoter sequence and initiates RNA synthesis de novo without the need of
any transcription factor.57 Due to its structural simplicity compared to larger multisubunit cellular
RNAPs, T7 RNA polymerase is an attractive target for structural and functional studies.69, 70 Its overall
structure was first determined by X-ray crystallography in 1993 by Sousa et al..69 Since then, a dozen
of T7 RNA polymerase structures in complex with its promoter, with its natural inhibitor T7
lysozyme, with alpha beta methylene ATP, bound to different DNA-RNA hybrids and at different
stages of transcription have been determined.57, 70-76 The structures revealed the similarities between the
three-dimensional shape of T7 RNAP and that of other nucleic acid polymerases with different
substrate specificities. T7 RNAP and other members of the DNA polymerase I family have modular
structures and are composed of thumb, palm and fingers subdomains resembling the shape of an open
right hand (Figure 1.5).69, 77 T7 RNAP is homologous to the RNA polymerases from the T3 and SP6
phages and additionally displays extensive sequence homology to the polymerase domain of Klenow
fragment of E. coli DNA polymerase I.58,
69, 77-79
The polymerase is organized around a deep cleft,
forming a U-shape fold that is characteristic for all pol I polymerases. This cleft can accommodate
double-stranded DNA to form the template binding site.
Figure 1.5: Overall structure of T7 RNA polymerase. Subdomains are coloured as follows: N-terminal (green),
thumb (blue), palm (cyan) and fingers (yellow). Lysozyme from the original structure was omitted (PDB:
1ARO).
8
1. Introduction
Additionally, T7 RNA polymerase contains different auxiliary domains and motifs that are
unique to phage type RNA polymerases and that fulfill specialized functions like promoter
recognition.77 These include the N-terminal domain and the specificity loop, both involved in the
promoter recognition process and melting of the DNA double-helix.71 The structure around the active
site in the palm domain is highly conserved and contains two catalytic aspartatic residues D812 and
D537 coordinating the two metal ions that are required during the phosphoryl transfer reaction.56, 80
Extensive structure-function studies revealed the tyrosine residue at amino acid position 639
responsible for discrimination of ribose versus deoxyribose substrates by formation of a hydrogen
bond with the 2’-OH group of the incoming ribonucleotide.3, 4, 81, 82 Amino acid H784 is supposed to be
involved in substrate selection during transcription initiation by interacting with the first incoming
nucleotide.57, 3, 83
T7 RNA polymerase performs transcription in an identical manner than bacterial and
eukaryotic multisubunit RNA polymerases.53,
69
To initiate RNA synthesis, T7 RNA polymerase must
recognize and bind to its specific promoter sequence (5’-TAATACGACTCACTATAGGGAGA-3’)
which is conserved from positions -17 to +6 relative to the transcription start site and which is located
upstream of the transcription initiation site. The double-stranded DNA helix is unwind and strands are
separated at a length of about 7-8 nucleotides to form the transcription bubble.84-86 RNA synthesis
begins preferentially with a guanosine at the transcription initiation site using the non-coding strand of
the DNA as template.57,
87
The efficiency of transcription initiation dramatically decreases when the
DNA-template codes for other nucleotides than guanosine at the two first positions.65, 88,
89
During the
early stages of transcription, T7 RNAP maintains the contact to the upstream promoter sequence while
the active site translocates downstream and starts with the synthesis of a short transcript to extend the
transcription bubble.90, 91 This so called initiation complex is unstable and highly sensitive for abortion
resulting in premature release of short RNA transcripts 2-9 nucleotides in length.92 88, 91 When reaching
a transcript length of about 8-10 nucleotides, the RNA polymerase undergoes a conformational change
to form a stable, processive elongation complex.73,
75
This transition state is a highly dynamic process
resulting in the release of the promoter region and displacement of the 5’-end of the nascent RNA
strand.93,
94
In the elongation phase, the RNA polymerase proceeds processively down the DNA
template, synthesizing a full-length RNA transcript. Termination of transcription takes place at
sequences that form GC-rich hairpins followed by runs of uridines.
9
1. Introduction
1.4
Directed evolution
1.4.1
General
Directed evolution of proteins is a powerful and widely used method in protein engineering to
evolve enzymes with properties not found in nature or to improve properties like activity, stability or
selectivity.95-99 It is an iterative process of random mutagenesis, expression of mutant enzymes and
high-throughput screening or selection for altered properties. The gene of the most improved variant is
then used as the template for further rounds of mutagenesis, expression and screening and the process
repeated until the desired level of improvement has been achieved (Figure 1.6). The step of random
mutagenesis can either address the entire target gene or selected amino acid positions. Techniques
commonly used in this step are error-prone PCR, saturation mutagenesis, spike mutagenesis, and DNA
shuffling.100-104 The use of mutator strains has also been reported to result in a reasonable
diversification of the target gene.105 The resulting diversified gene library is afterwards transferred into
a host organism e.g. E. coli for protein expression. In order to have a direct correlation between
genotype and enzyme properties, host cells containing mutants of the library have to be separated from
each other.106, 107 In high-throughput screening this is accomplished by a compartmentalization step in
multiwell plates. The generation of discrete compartments formed by a water-in-oil emulsion or
simple growth of host cells on plates containing a selection marker has been reported for different
selection strategies.2,
107
Advantages of directed protein evolution over other protein engineering
methods like the structure-based site-specific mutagenesis, also referred to as rational design, is that no
structural or mechanistic information is required that often derive from X-ray crystallographic data.
Whatever technique is used for mutagenesis, an adequate high-throughput screen or selection method
is required.
Figure 1.6: General scheme depicting the continuous cycle of directed protein evolution.
10
1. Introduction
1.4.2
Directed evolution of nucleic acid polymerases
DNA and RNA polymerase are interesting target enzymes for directed evolution. DNA
polymerases are used in a wide range of biotechnological applications like PCR, PCR-based methods
and DNA sequencing including next generation sequencing.108-110 RNA polymerases are commonly
used for in vitro transcription and also in SELEX.65,
66, 111, 112
Due to the continuous improvement of
these techniques and for the development of new polymerase-based techniques, there is a high demand
for DNA and RNA polymerases with altered properties like increased fidelity, thermostability or
increased substrate spectra. Methods of directed evolution have already successfully been used to
evolve DNA polymerases with increased substrate like the ability to incorporate ribonucleotides,113-115
modified nucleotides116-119 or nucleotides of unnatural DNA analogues.120,
121
DNA polymerases with
increases reverse transcriptase122 or lesion-bypass activity123 and with enhanced fidelity in mismatch
extension124,
125
have also been successfully evolved. Recent progress in this field includes three
examples of DNA polymerase evolution performed by Holliger and coworkers. They evolved DNA
polymerase variants that can efficiently incorporate and extend hydrophobic base analogues126,
synthesize and reverse transcribe six synthetic genetic polymers120 and efficiently synthesize RNA as
well as fully pseudouridine-, 5-methyl-C-, 2’-fluoro-, or 2’-azido-modified RNAs primed from a wide
range of primer chemistries comprising DNA, RNA, locked nucleic acid (LNA), or 2’-O-methylDNA, respectively.127 It is worth to mention that the above given examples do not cover all efforts that
have been done in this field.
Both, selection and screening methods have been established and proven to be suitable for the
directed evolution of DNA polymerases.128,
129
Selection methods include phage display130,
115
and
compartmentalized self-replication (CSR), a method conducted in compartments of a heat-stable
water-in-oil emulsion based on a simple feedback loop.131 This loop involves a step in which the
polymerase has to replicate its own encoding gene resulting in the enrichment of active variants that
can further be selected. Other selection methods based on such activity feedback loops had been
developed and successfully used by Loeb and coworkers to evolve DNA polymerase mutants with
increased fidelity compared to the wildtype enzyme.132 For this purpose, the authors used a reporter
plasmid that contained an antibiotic resistance gene including an opal stop codon that had to be
restored for cell survival. The same authors also reported the development of a genetic
complementation system in which DNA polymerase I-deficient E. coli cells were used to select active
DNA polymerase variants from different libraries.133 The fact that DNA and RNA polymerases are
directly involved in processes required for cell survival of the host organism enables the development
of such in vivo selection methods that are well suited to pre-screen a mutant library for active variants.
In addition to those selection methods, different screening approaches including nucleotide
incorporation assays134, primer-extension reactions125 or PCR124 have been established. The formation
of reaction products can directly be detected by fluorescence readout using DNA double strand
binding dyes such as SYBR Green I.125
11
1. Introduction
In contrast to DNA polymerases, only a few approaches have been reported on the directed
evolution of RNA polymerases so far. Due to its stringent promoter specificity, robust activity, and
widespread application, T7 RNA polymerase is an interesting target for protein engineering. By using
directed protein evolution T7 RNA polymerase variants with increased acceptance of 2’-O-methyl (2’OCH3)-modified nucleotides,2 altered promoter specificities135, 136 or error-prone polymerase activity137
have been generated, respectively. The directed evolution of a T7 RNA polymerase with increased
acceptance of 2’-OCH3-modified nucleotides has proven this methodology powerful for example for
used of the evolved RNA polymerases for SELEX involving 2’-modified nucleic acids.138
In all of the reported approaches the authors used in vivo selection methods including a
feedback loop for selection of T7 RNA polymerase variants with desired properties. Chelliserrykattil
et al. used an activity-based method for selecting functional T7 RNA polymerase variants based on the
ability of a T7 RNA polymerase to reproduce itself.135 In this system, the T7 RNA polymerase library
was cloned under the control of its own promoter that contained mutations to create a so called
‘autogene’. Active variants that were able to recognize the mutated promoter generated more mRNA
which was isolated, reverse transcribed and amplified by PCR. The so obtained genes were re-cloned
and re-transformated to enter additional rounds of selection to accumulate polymerase variants with
altered promoter specificities. Later on, the same authors reported on a modified version of this screen
in which the polymerase not only had to reproduce itself, but also had to transcribe an antibiotic
resistance element (chloramphenicol acetyl transferase, CAT) required for cellular survival.2 After
growing on plates supplemented with chloramphenicol, polymerase variants which were active enough
to produce sufficient CAT survived the selection and had further been tested for an increased
acceptance of a variety of 2’-modified NTPs. Recently, another group reported a system called phageassisted continuous evolution (PACE) enabling the continuous directed evolution of gene encoded
molecules which can be linked to protein production in E. coli.139 Using this methodology, T7 RNA
polymerase variants had been evolved that recognize a distinct promoter and initiate transcription with
ATP and CTP instead of GTP.
1.5
Modified RNA and its applications
1.5.1
Overview
Chemical modifications of RNA are used to alter the biochemical and biophysical properties
of RNA molecules in order to use them in nucleic acids based technologies such as antisense
oligonucleotides140, short interfering RNA (siRNA)141,
142
or as aptamers.143-146 Furthermore, modified
RNA is used to study RNA structure and dynamics by spectroscopic methods or X-ray
crystallography.147 The overall number of available chemical RNA modifications is impressive and
only some of them will be mentioned here. Unmodified RNA is inherently unstable due to its 2’-
12
1. Introduction
hydroxyl group that promotes strand cleavage by alkaline hydrolysis or ribonucleases. For use of RNA
as therapeutic agent in the above mentioned key technologies, an increased metabolic stability is a
fundamental requirement. Chemically modified siRNA is well suited as a therapeutic since it has been
proven to exhibit an enhanced nuclease resistance, to decrease off-target effects and immune
activation and to have some other advantageous properties like improved pharmacokinetics and dynamics.148-150 Similar positive effects have been reported for chemically modified antisense
oligonucleotides that exhibit higher binding affinities compared to their unmodified counterparts.140
Modifications have been introduced at different positions like nucleobase, backbone, sugar and
terminus.150,
151
However, modifications at the 2’-position of the ribose sugar have proven to be most
successful and have the advantage that they can be incorporated in both purines and pyrimidine
nucleosides thus can be site-specifically positioned within the RNA. Additionally, 2’-modifications
are known to increase the chemical stability and nuclease resistance.152-154 2’-O-Methyl (2’-OCH3) and
2’-fluoro (2’-F) modifications are commonly used in siRNA techniques since they are very well
tolerated in the guide strand resulting in enhanced performance of the modified siRNA.155-157
Recently, 2’-azido (2’-N3) modifications were evaluated for their potential as siRNA and the modified
RNA oligonucleotides showed efficient silence activity.158 2’-modifications are also attractive for the
idenification of stable high affinity RNA aptamers by SELEX.159, 160 Pegabtanib, a 2’-F pyrimidine and
2’-OCH3 purine substituted anti-VEGF aptamer that was evolved using SELEX is in clinical use for
age-related macular degeneration (AMD).161 Modified RNA is also used in techniques for the
investigation of RNA structure and conformational dynamics. Many of these techniques rely on the
availability of site-specifically modified RNA. Spectroscopic methods like nuclear magnetic
resonance (NMR) spectroscopy or electron paramagnetic resonance (EPR) spectroscopy require
selectively isotopic labeled RNA by use of
13
C,
15
N or
19
F-labeled building blocks or spin-labeled
RNA, respectively.147 Modern techniques for phase determination in X-ray crystallography such as
MAD (multiple anomalous dispersion) depend on heavy metal modifications within the RNA by using
5-iodo and 5-bromo pyrimidines or selenium-modified ribonucleosides.162 Beside the advantageous
and well explored 2’-methylseleno (2’-SeCH3) modification,163,
164
ribonucleosides in which the 4’-
oxygen of the ribose is replaced by selenium can be incorporated into RNA oligonucleotides by solidphase
synthesis
using
4’-Se-uridine
and
4’-Se-5-methyl-uridine.165,
166
Furthermore,
phosphoroselenoate-modified RNA has been synthesized enzymatical using T7 RNA polymerase.167
13
1. Introduction
1.5.2
Synthesis of modified RNA
In general, modified RNA is available by two approaches: chemical solid-phase synthesis
using phosphoramidite building-blocks and enzymatic synthesis using ribonucleoside triphosphates
and RNA polymerases. Solid-phase synthesis offers the advantage of site-specific positioning of the
desired modification within the target RNA. However, the synthesis on solid support suffers from
some limitations like the oligonucleotide length and degree of modification. RNA synthesis is still
limited to 50-80 nucleotides in length, depending on the type of modification and the used chemical
strategy.168,
169
Additionally, some modified phosphoramidite building blocks exhibit only moderate
coupling efficiencies compared to the unmodified phosphoramidites. Methods to overcome the length
limitations of the synthesis on solid-sopport include the enzymatic ligation of modied and unmodified
RNA fragments.169
The enzymatic synthesis of modified RNA is commonly performed by in vitro transcription
using the DNA-dependent RNA polymerases of certain phages like T7 RNA polymerase and the
respective modified NTPs.65,
66
The enzymatic approach represents a powerful alternative to the
chemical synthesis because larger modified RNA molecules can be synthesized. However, depending
on the modification these modified nucleotides are often poor substrates at full substitution. 2’Modified nucleotides are inefficiently incorporated using T7 RNA polymerase wildtype.170 Based on
structural analysis and mutagenesis, mutants have been generated that exhibit an increased acceptance
for a variety of 2’-modified nucleotides.
1, 2, 171
Among these, the double mutant Y639F, H784A
reported by Sousa and coworkers is the only one that has been successfully used for the synthesis of
2’-OCH3 modified versatile aptamers.159,
160
Transcription using T7 RNA polymerase does not
necessarily start with the incorporation of a 5’-triphosphate, but also nucleotides with modifications
attached to the 5’-position are well tolerated. For this reason, the use of so-called initiator nucleotides
that have modifications attached to the 5’-position and thus resulting in 5’-terminal modified RNA is a
widespread technique for bioconjugation.172-175
14
2. Aim of this Work
2 Aim of this Work
The main goal of this work was the identification of a T7 RNA polymerase mutant that is able
to incorporate 2’-methylseleno (2’-SeCH3)-modified nucleotides into RNA. 2’-SeCH3-modified RNA
is used in X-ray crystallography for the determination of RNA structures. The enzymatic synthesis of
2’-SeCH3-modified RNA using an RNA polymerase should provide a synthetic alternative to
commonly used chemical solid-phase synthesis for the preparation of long modified RNA sequences.
In order to identify a T7 RNA polymerase that can be used for the enzymatic synthesis of 2’-SeCH3modified RNA, two different approaches were followed. In the first approach, it should be
investigated whether known T7 RNA polymerase mutants with reduced discrimination between noncanonical and canonical nucleotides are able to incorporate 2’-SeCH3-modified nucleotides into RNA.
In the second approach, a T7 RNA polymerase mutant with an increased acceptance of 2’-SeCH3NTPs should be evolved by directed protein evolution. For this purpose, different T7 RNA polymerase
mutant libraries should be constructed and screened for active variants with increased acceptance of
2’-SeCH3-NTPs. A screening assay based on fluorescence readout for the detection of active T7 RNA
polymerase variants should be established and used to screen the mutant libraries in high-throughput.
Finally, the identified mutants should be characterized in order to draw conclusions about the
contribution of each amino acid substitution to the increased substrate spectra. The characterization
should thus provide further insights into the complex processes of substrate recognition and nucleotide
discrimination of T7 RNA polymerase.
In the second part of this work, the DNA-dependent enzymatic synthesis of unnatural nucleic
acid polymers, called xeno-nucleic acids (XNA), should be investigated. By using a selection of DNA
polymerases and mutants of eukaryotic, prokaryotic, and archaic origin from different evolutionary
families, it should be tested whether the used DNA polymerases show a propensity to polymerize a
variety of sugar-modified nucleotides in primer extension experiments. A focus should be done on the
enzymatic synthesis of deoxyxylose nucleic acids (dXNA), a potential orthogonal nucleic acid
candidate. It should be explored whether the used DNA polymerases are able to synthesize
deoxyxylose nucleic acids in order to identify a promising candidate to evolve into an efficient DNAdependent dXNA polymerase by directed protein evolution.
15
16
3. Results and Discussion
3 Results and Discussion
3.1
Enzymatic Synthesis of 2’-Selenium-modified RNA Using Engineered
RNA Polymerases
3.1.1
Introduction
The large number of highly relevant non-coding RNA sequences and the growing interest into
their functions demands a fast and powerful method for their structural characterization. Along with
NMR spectroscopy, X-ray crystallography is the method of choice for nucleic acid structure
determination.176, 177 Once crystals of a novel RNA have been grown and diffraction patterns recorded,
phasing of the data can be a serious challenge. Modern techniques to facilitate phase determination
have been developed that are depending on anomalous scattering centers within the respective RNA
crystal. There are several approaches available to introduce these scattering centers. Heavy-atom
derivatization of nucleic acids can be achieved by soaking of the RNA crystal in salt solutions, cocrystallization with heavy metal ions or by covalent modification with halogen atoms using 5-iodo and
5-bromo pyrimidines.178, 179 One drawback of halogen modified oligonucleotides is that they are highly
photo-reactive species and their damage during X-ray radiation has been reported as a serious
limitation.180
Selenium derivatization of nucleic acids represents a powerful alternative to conventional
heavy-atom derivatization for structure determination using the multiwavelength anomalous
dispersion technique (MAD).181,
182
For the modification of RNA with selenium several positions are
amenable, although only one, namely the 2’-methylseleno (2’-SeCH3) modification has achieved
prominent applications in X-ray structure analysis. The introduction of selenium at the ribose 2’position of nucleotides, oligonucleotides and larger RNAs was pioneered by Huang and Micura and
their respective coworkers.163, 183 Structural studies using the 2’-methylseleno-modified RNA sequence
in comparison to the unmodified RNA sequence demonstrated that for mostly all of the tested RNAs
this modification did not affect the crystallization conditions and appeared to have little effect on the
stability and structure.184 Additionally, 2’-SeCH3-modified RNA showed an increased resistance
towards X-ray-induced radiation damage compared to commonly used 5-halogen-RNA and is
therefore an attractive alternative for derivatization.164 As proof of concept, Micura et al. have used 2’SeCH3-modified RNA for the structure determination of the Diels-Alder ribozyme and of HIV-1
genomic RNA dimerization initiation site (DIS) extended duplex in complex with several
aminoglycoside antibiotics.185, 186
17
3. Results and Discussion
2’-SeCH3-modified RNA (Figure 3.1 A) is accessible by chemical solid-phase synthesis using
phosphoramidite building blocks. A toolbox of all four 2’-SeCH3 nucleoside phosphoramidites is
available and thus permits the possibility of site-specific positioning of the modification within the
RNA target sequence. However, the chemical synthesis of 2’-SeCH3-RNA is limited to about 50
nucleotides in length due to the inherent limitations of RNA synthesis on solid support. The synthesis
of 2’-SeCH3-modified RNA of sizes up to about 100 nucleotides can only be accomplished in
combination with enzymatic ligation techniques.187,
188
Both preparation techniques are laborious and
along with the size restriction they can be seen as a severe limitation to widespread the method for the
structure determination of novel non-coding RNA sequences of sizes between 50 and 200 nucleotides.
In order to overcome these drawbacks, the enzymatic synthesis of 2’-SeCH3-RNA using RNA
polymerases along with the 2’-SeCH3-modified ribonucleoside 5’-triphosphates (2’-SeCH3-NTPs)
(Figure 3.1 B) represents a promising approach. For this purpose, a synthetic route towards all four
2’-SeCH3-NTPs was elaborated in the group of Prof. Ronald Micura from the University of Innsbruck
in Austria.
Figure 3.1: A) Chemical structure of 2’-methylseleno RNA; B) Chemical structure of 2’-methylseleno NTPs.
The synthesis of larger RNA targets beyond the length limitations of chemical solid-phase
synthesis is commonly accomplished by in vitro transcription using T7 RNA polymerase.65 However,
modified nucleotides are not natural substrates for T7 RNA polymerase wildtype and are thus often
poorly accepted during transcription. Padilla et al. have used rational design to generate a T7 RNA
polymerase mutant with increased acceptance of 2’-F and 2’-NH2 modified triphosphates.171 This
mutant had been generated by replacing tyrosine at position 639 in the nucleotide recognition site with
the less bulky amino acid phenylalanine (Y639F). Nevertheless, nucleotides with larger substituents
such as 2’-OCH3 were only poor substrates for this mutant. The same authors have later reported a
second T7 RNA polymerase mutant that had been generated by the introduction of a second mutation
at position 784 (H784A). This T7 RNA polymerase double mutant (Y639F, H784A) exhibits
markedly increased incorporation efficiencies for nucleotides with bulky 2’-substituents like 2’-OCH3
18
3. Results and Discussion
and 2’-N3.1 Recently, Chelliserrykattil et al. have demonstrated that directed evolution is a promising
strategy to evolve T7 RNA polymerase mutants with increased acceptance of 2’-modified nucleotides.
By mutating certain positions in the polymerase wildtype gene and subsequent selection of active
variants from a mutant library, the authors were able to evolve a mutant (E593G, Y639V, V685A,
H784G) that exhibits an increased acceptance of 2’-OCH3-ATP, -CTP, and -UTP compared to T7
RNA polymerase wildtype.2
Based on these previous findings, it should be tested in this work whether the known T7 RNA
polymerase mutants with increased tolerance towards 2’-modified nucleotides exhibit also an
increased acceptance of the synthesized 2’-SeCH3-NTPs. Therefore, the T7 RNA polymerase mutants
M1 (Y639F, H784A) and M2 (E593G, Y639V, V685A, H784G) were generated by site-directed
mutagenesis starting from the T7 RNA polymerase wildtype gene. The mutants as well as the wildtype
T7 RNA polymerase were expressed in E. coli, purified via their N-terminal polyhistidine affinity tags
and afterwards tested in transcription reactions for incorporation of the 2’-SeCH3-modified
nucleotides. Different RNA target sequences with known crystallization behaviour have been chosen
to be transcribed in the presence of 2’-SeCH3-NTPs as proof of principle. In addition, T7 RNA
polymerase mutants with increased acceptance of 2’-SeCH3-NTPs should be evolved by directed
protein evolution. Starting from the T7 RNA polymerase wildtype gene, a mutant library should be
constructed by saturation mutagenesis of amino acid positions Y639 and H784. A second mutant
library should be constructed starting from the gene of T7 RNA polymerase mutant M2 by random
mutagenesis using error-prone PCR, a method well established in our laboratory to address non
predictable amino acid positions. To screen the T7 RNA polymerase mutant libraries in highthroughput, a screening assay based on fluorescence readout of synthesized RNA after in vitro
transcription should be established for the identification of active variants from the libraries. The use
of fluorescence readout based screening assays had already been reported for the directed evolution of
DNA polymerases with increased substrate spectra.122, 125 Active hits from both libraries should further
be tested for an increased acceptance of the 2’-SeCH3-NTPs in
32
P-based in vitro transcription
ractions. The characterization of those T7 RNA polymerase variants should thus provide further
insights into the complex process of substrate recognition of 2’-modified nucleotides.
19
3. Results and Discussion
3.1.2
Construction of T7 RNA polymerase wildtype recombinant plasmid for protein
expression in E. coli
The gene of T7 RNA polymerase wildtype was purchased from Geneart. The nucleotide
sequence was optimized for protein expression in E. coli and the sequence was designed to contain an
N-terminal Factor Xa protease cleavage site for removal of the vector coded N-terminal polyhistidine
tag (6×His-tag) from the protein. The gene was cloned into the expression vector pGDR11 using SphI
and HindIII restriction sites. The pGDR11 vector is a derivative of the pQE31 vector containing an
additional lacIq gene coding for the lac repressor protein to control basal expression.189 The pGDR11
vector is suitable for protein expression in E. coli under control of an IPTG inducible T5 promotor and
provides the target protein with an N-terminal 6×His-tag for protein purification using immobilized
metal ion affinity chromatography. The resulting construct T7 RNAP wildtype-pGDR11 was used for
the expression of T7 RNA polymerase wildtype and served as a starting point for the generation of T7
RNA polymerase mutants and mutant libraries.
3.1.3
Generation of T7 RNA polymerase mutants M1 (Y639F, H784A) and M2
(E593G, Y639V, V685A, H784G)
T7 RNA polymerase mutant M1 (Y639F, H784A) was generated in two steps by site-directed
mutagenesis starting from the construct T7 RNAP wildtype-pGDR11. T7 RNA polymerase mutant
M2 (E593G, Y639V, V685A, H784G) was generated by using a QuikChange Multi Site-Directed
Mutagenesis Kit, where all four mutations have been introduced simultaneously starting from the
construct T7 RNAP wildtype-pGDR11. Sequences were confirmed after each step of mutagenesis by
DNA sequencing. E. coli BL21-Gold (DE3) cells and E. coli XL10-Gold cells were transformed with
the resulting constructs for protein expression and long term storage at -80°C, respectively.
3.1.4
Heterologous expression and purification of T7 RNA polymerases in E. coli
BL21-Gold (DE3)
Protein expression of T7 RNA polymerases was conducted using E. coli BL21-Gold (DE3)
cells containing the constructs T7 RNAP wildtype-pGDR11, T7 RNAP M1-pGDR11, and T7 RNAP
M2-pGDR11, respectively. Cells were cultivated in LB-medium supplemented with the selection
marker carbenicillin and proteins expressed for 4 h at 37°C after the induction with IPTG. SDS-PAGE
gel analysis indicated the expression of T7 RNA polymerases which possess a molecular weight of
about 101 kDa (Figure 3.2 A).
20
3. Results and Discussion
Figure 3.2: Expression and purification of T7 RNA polymerases. (A) SDS-PAGE gel analysis of the expression
of T7 RNA polymerase mutant M2 in E. coli BL21-GOLD(DE3) cells. M: Marker; t0: Sample before induction
with IPTG; t240: Sample 4 h after the induction with IPTG. Samples were adjusted to the same cell density prior
to loading. (B) SDS-PAGE gel analysis of purified T7 RNA polymerase wildtype (wt), T7 RNA polymerase
mutant Y639F, H784A (M1) and T7 RNA polymerase mutant E593G, Y639V, V685A, H784G (M2). Proteins
were adjusted to the same concentration.
T7 RNA polymerases containing an N-terminal 6×His-tag were purified by immobilized metal
ion affinity chromatography using Ni-IDA chelating sepharose with an ÄKTA purifier
chromatography system. SDS-PAGE gel analysis indicated that the purified proteins were ≥95% pure
after this purification step. Protein concentrations were determined via Bradford protein assay and
adjusted to the same value for further investigations (Figure 3.2 B).
3.1.5
In vitro transcription of relevant RNA sequences using natural NTPs and 2’SeCH3-UTP
As a proof of principle, it was first tested whether the T7 RNA polymerase mutants M1 and
M2, or even the T7 RNA polymerase wildtype, are able to synthesize 2’-SeCH3-modified RNA of
target sequences with known crystallization behaviours. For this purpose, a 27 nucleotides long RNA
sequence had been chosen first that mimics the sarcin-ricin loop (SRL) domain of the E. coli 23S
rRNA. The SRL is a well studied RNA motif which has been successfully crystallized with various
mutations and modifications.164,
190
2647–2673 in E. coli 23S rRNA.
The used 27-mer RNA represents the nucleotides at positions
32
P-based in vitro transcription assays were performed using the
purified T7 RNA polymerase mutants and T7 RNA polymerase wildtype. The reaction products were
analyzed by denaturing PAGE and phosphorimaging. Reactions were performed with all four natural
NTPs, with 2’-SeCH3-UTP replacing UTP and without UTP. It was observed that all of the three
21
3. Results and Discussion
tested T7 RNA polymerases had problems with the synthesis of the short 27-mer RNA sequence. A
full length transcript was only observed in reactions with NTPs using T7 RNA polymerase wildtype
and mutant M2, whereas the amount of RNA synthesized by mutant M2 was poor. No full length
product was observed for any of the three tested RNA polymerases in reactions with 2’-SeCH3-UTP or
in the control reactions without UTP (Figure 3.3).
Figure 3.3: In vitro transcription of the 27 nt E. coli sarcin-ricin loop RNA sequence. Reactions were conducted
in the presence of NTPs (+UTP), in the presence of ATP, GTP, CTP and 2’-SeCH3-UTP (+2’-SeMe-UTP) and
in the presence of ATP, GTP, and CTP without UTP (-UTP). M: 32P-labeled 30-mer marker RNA
oligonucleotide; 1, 4, 7: Reactions performed with T7 RNA polymerase wildtype; 2, 5, 8: Reactions performed
with T7 RNA polymerase mutant M1; 3, 6, 9: Reactions performed with T7 RNA polymerase mutant M2.
Reaction products were body-labeled by the addition of [α-32P]GTP and visualized by denaturing PAGE and
phosphorimaging.
In order to investigate whether the purified T7 RNA polymerase mutants could generally be
used to synthesize short RNA target sequences, the enzymatic synthesis of different relevant RNA
sequences was investigated in the presence of natural NTPs. For this purpose, two DNA templates
coding for the 27-mer sarcin-ricin loop mimic with additional nucleotides at both ends were used.
These additional nucleotides were added in order to ease the enzymatic synthesis when using T7 RNA
polymerase due to purines at the 5’-end of the RNA transcript known to be required for efficient
transcription initiation. Those RNA sequences reproduce the nucleotides at positions 2644–2676 (33mer) and 2643–2673 (35-mer) in E. coli 23S rRNA. Additionally, the enzymatic synthesis of another
27-mer RNA sequence was investigated: the HIV-1 TAR hairpin.191 DNA templates with a double-
22
3. Results and Discussion
stranded promoter region were formed by assembling the synthetic oligonucleotides with the T7
promotor sequence in reaction buffer prior to transcription. In vitro transcription assays using the three
T7 RNA polymerases were performed in the presence of NTPs and the reaction products analyzed by
denaturing PAGE and phosphorimaging. In doing so, it was observed that the T7 RNA polymerase
mutants M1 and M2 were not able to synthesize any of the desired unmodified RNA sequences
whereas T7 RNA polymerase wildtype was able to synthesize full-length products for all three targets.
PAGE gel analysis also indicated that the yield of synthesized RNA in case of the 27 nucleotides
sarcin-ricin loop by T7 RNA polymerase wildtype is poor in comparison to the yield of the other RNA
sequences (Figure 3.4).
Figure 3.4: In vitro transcription of different RNA target sequences. All reactions were conducted in the
presence of natural NTPs. M: 32P-labeled 30-mer marker RNA oligonucleotide; 1: Reactions performed with T7
RNA polymerase wildtype; 2: Reactions performed with T7 RNA polymerase mutant M1; 3: Reactions
performed with T7 RNA polymerase mutant M2. Reaction products were body-labeled by the addition of [α32
P]GTP and visualized by denaturing PAGE and phosphorimaging.
23
3. Results and Discussion
In another attempt, the ability of the T7 RNA polymerase mutants to synthesize a relevant
RNA sequence was investigated by use of a DNA template coding for a 29-mer RNA fragment
deriving from helix 6 of the human signal recognition particle (hSRP). The DNA template was
designed to provide the RNA target sequence with a hammerhead ribozyme motif at the 3’-end which
is cleaved off from the target sequence co-transcriptionally. This RNA sequence had been successfully
synthesized by in vitro transcription using T7 RNA polymerase wildtype in a sufficient amount for
structure determination by Wild et al..192 The DNA template for in vitro transcription was prepared by
PCR as it had been reported that the use of double-stranded transcription templates yields in more
RNA compared to single-stranded synthetic DNA oligonucleotides with a double-stranded promoter
region.159 Transcription reactions were performed in the presence of all four natural NTPs, in the
presence of ATP, CTP, GTP, and 2’-SeCH3-UTP and without UTP as control reaction. Activities
obtained with T7 RNA polymerase wildtype were compared to those obtained with the mutants M1
and M2. The concentration of Mg2+-ions in the reaction buffer was increased from 6 mM to 10 mM for
efficient ribozyme cleavage. Reaction products were body-labeled by the addition of [α-32P]GTP and
analyzed by denaturing PAGE and phosphorimaging. It was observed that T7 RNA polymerase
wildtype was able to synthesize the 78 nucleotides long full-length transcripts in reactions with NTPs
and that the ribozyme was cleaved off from the target sequence resulting in the desired 29-mer RNA
sequence in reasonable yields. However, in reactions in which UTP was replaced by 2’-SeCH3-UTP
and in the control reaction without UTP no reaction products were formed. In reactions with T7 RNA
polymerase mutants M1 and M2, only small amounts of full-length transcripts and of the cleaved-off
target RNA sequence were observerd in reactions with NTPs. Reactions with 2’-SeCH3-UTP instead
of natural UTP resulted in marginal amounts of the uncleaved full-length transcripts indicating the
incorporation of the 2’-SeCH3-modification. In control reaction without UTP no reaction products
were observed (Figure 3.5).
24
3. Results and Discussion
Figure 3.5: In vitro transcription of the 29 nt human SRP-RNA attached to a hammerhead ribozyme. Left:
Denaturing PAGE gel. M: 32P-labeled 30-mer marker RNA oligonucleotide; 1: Reactions performed in the
presence of natural NTPs (+UTP); 2: Reactions performed in the presence of ATP, GTP, CTP, and 2’-SeCH3UTP (+2’-SeCH3-UTP); 3: Reactions performed in the presence of ATP, GTP, and CTP but without UTP (UTP). Reactions of T7 RNA polymerase wildtype (wt) were compared to reactions with T7 RNA polymerase
mutant M1 and M2. Right: Sequence and partially secondary structure depiction of the 78 nt full-length
transcript. The sequence of the desired 29-mer RNA target is indicated in red. The arrow indicates the ribozyme
cleavage site.
3.1.6
Synthesis of 2’-SeCH3-modifed RNA by T7 RNA polymerase mutants M1 and
M2
After these initial experiments to synthesize a variety of short RNA sequences by using the T7
RNA polymerase mutants M1 and M2, the enzymatic synthesis of 2’-SeCH3-modified RNA was
investigated by using a longer RNA target sequence.
32
P-based in vitro transcription assays were
conducted using a double-stranded DNA template including the T7 promotor sequence that coded for
an 89-mer RNA transcript. This DNA template was designed to code for a purine-rich sequence at the
5’-end of the RNA transcript that is well known to stabilize the initiation complex during transcription.
Thereby, the designed template enabled the T7 RNA polymerase mutants M1 and M2 to synthesize
RNA in amounts comparable to those achieved by T7 RNA polymerase wildtype in reactions with
25
3. Results and Discussion
natural NTPs (Figure 3.6, lane 1). As reported in the literature, T7 RNA polymerase mutant M2 was
observed to be less efficient with natural NTPs than the wildtype polymerase.2 To investigate the
acceptance of 2’-SeCH3-UTP by the T7 RNA polymerases, transcription reactions in which UTP was
replaced by 2’-SeCH3-UTP in the presence of ATP, GTP, and CTP were performed. In doing so, it
was observed that both T7 RNA polymerase mutants M1 and M2 were able to synthesize full-length
transcripts whereas the wildtype T7 RNA polymerase fails to synthesize any detectable transcription
product (Figure 3.6 A, lane 2). In control reactions without UTP, no full-length transcripts were
detected for any of the tested T7 RNA polymerases (Figure 3.6 A, lane 3). This observation led to the
assumption that formation of the full-length products in reactions with 2’-SeCH3-UTP resulted from
the incorporation of 2’-SeCH3-modified nucleotides into RNA. When T7 RNA polymerase mutant M1
was used to incorporate the 2’-SeCH3-modification into RNA, it was observed that the polymerase
stalled when incorporating two consecutive 2’-SeCH3-modified nucleotides leading to shorter abortive
transcripts of about 77 nucleotides in size (Figure 3.6 A, M1, lane 2).
Figure 3.6: Enzymatic synthesis of 2’-SeCH3-modified RNA. PAGE gel analysis of transcription reactions
comparing T7 RNA polymerase wild-type (wt) with mutant M1 (M1) and mutant M2 (M2) after an incubation
time of 2 h at 37 °C. The sequences of the 89 nt RNA transcripts are depicted above each gel. A) 1: Reactions
with all four NTPs (+UTP); 2: Reactions in which UTP was replaced by 2’-SeCH3-UTP (+2’-SeCH3-UTP); 3:
Control reactions without UTP but with ATP, GTP, and CTP (-UTP). B) 1: Reactions with all four NTPs
(+CTP); 2: Reactions in which CTP was replaced by 2’-SeCH3-CTP (+2’-SeCH3-CTP); 3: Control reactions
without CTP but with ATP, GTP, and UTP (-CTP).
26
3. Results and Discussion
Next, the acceptance of 2’-SeCH3-CTP by the T7 RNA polymerases was investigated.
32
P-
based transcription reactions were conducted with 2’-SeCH3-CTP replacing CTP in the presence of
ATP, GTP, and UTP. The DNA template used in these reactions coded for an 89 nucleotides long
transcript where all U positions were changed to C and vice versa to guarantee comparable results as
obtained for reactions with 2’-SeCH3-UTP. Denaturing PAGE gel analysis revealed 2’-SeCH3-CTP as
a substrate for the T7 RNA polymerase mutants M1 and M2. Both mutants were able to synthesize the
2’-SeCH3-modified desired full-length transcripts in the presence of 2’-SeCH3-CTP (Figure 3.6. B,
M1 and M2, lane 2). In reactions with T7 RNA polymerase wildtype no full-length transcripts could
be detected demonstrating that 2’-SeCH3-CTP is not a substrate for the wildtype polymerase (Figure
3.6. B, wt, lane 2). In reactions with the four natural NTPs, all of the three tested T7 RNA polymerases
were able to synthesize unmodified full-length transcripts (Figure 3.6. B, lane 1). As already seen in
reactions with 2’-SeCH3-UTP, T7 RNA polymerase mutant M1 stalled at position 77 where two
consecutive 2’-SeCH3-modifications had to be incorporated resulting in a truncated transcript (Figure
3.6. B, M1, lane 2). In control reactions without CTP, it was observed that T7 RNA polymerase
mutant M1 was also synthesizing small amounts of full-length transcripts revealing that this mutant is
less accurate on the provided template (Figure 3.6. B, M1, lane 3).
To complete the picture, the acceptance of the 2’-SeCH3-modified purines by T7 RNA
polymerase was investigated. Therefore, transcription assays including 2’-SeCH3-ATP and 2’-SeCH3GTP were conducted. For the incorporation experiments involving 2’-SeCH3-ATP, a double-stranded
DNA template that coded for an 89-mer transcript in which 12 adenosine nucleotides had to be
incorporated was used. For the incorporation experiments involving 2’-SeCH3-GTP, the same doublestranded DNA template was used as for the incorporation experiments with 2’-SeCH3-UTP. This DNA
template coded for an 89-mer transcript in which 26 guanosines had to be incorporated, the first ones
at positions +1 to +3. Unfortunately, no full- length transcripts could be detected in reactions with the
2’-SeCH3-modified purines, neither in reactions with T7 RNA polymerase wildtype nor in reactions
with the mutants M1 and M2. The formation of any shorter truncated transcripts that might have
included the 2’-SeCH3-modification was not analyzed.
As PAGE gel analysis indicated, T7 RNA polymerase mutant M2 was most efficiently
incorporating 2’-SeCH3-modified nucleotides into RNA when 2’-SeCH3-UTP was provided as
substrate. To gain further insights into the incorporation efficiencies of the 2’-SeCH3-modification into
RNA, a time course experiment comparing T7 RNA polymerase wildtype with the mutants M1 and
M2 was performed. Transcription reactions were accomplished with 2’-SeCH3-UTP replacing UTP in
the presence of ATP, GTP, and CTP. Aliquots were withdrawn from the reaction mixtures and
quenched at distinct time points. In doing so, the previous observation that T7 RNA polymerase
mutant M2 is most efficiently incorporating the 2’-SeCH3-modified nucleotides into RNA was
confirmed. T7 RNA polymerase wildtype was not able to synthesize any detectable modified full-
27
3. Results and Discussion
length transcript. Nevertheless, T7 RNA polymerase wildtype was able to synthesize full-length
transcripts in the reaction with natural NTPs (Figure 3.7).
Figure 3.7: Time-course experiment for the enzymatic synthesis of 2’-SeCH3-modified RNA using T7 RNA
polymerase mutants and 2’-SeCH3-UTP. The PAGE gel shows transcription reactions using T7 RNA
polymerase wild-type (wt), mutant M1 (M1) and mutant M2 (M2). Aliquots of the reaction mixtures were
withdrawn at corresponding time points between 0.5 and 120 min and quenched with an equal volume of stop
solution containing 50 mM EDTA.
By comparing the total intensities of the formed full-length transcripts, the amount of
synthesized 2’-SeCH3-modified RNA formed by T7 RNA polymerase mutants M1 and M2 was
quantified. The graphic depiction demonstrates the efficiencies of both T7 RNA polymerase mutants
while incorporating the 2’-SeCH3-modification into RNA (Figure 3.8).
Figure 3.8: Quantification of the time-course experiment for the enzymatic synthesis of 2’-SeCH3-modified
RNA using T7 RNA polymerase mutants and 2’-SeCH3-UTP.
28
3. Results and Discussion
To determine the total amount of 2’-SeCH3-modified RNA that can be synthesized by using
T7 RNA polymerase mutant M2, large scale transcription assays were performed and the synthesized
RNA quantified. For this purpose, the full-length RNA was purified by denaturing PAGE after
transcription to remove all truncated RNA transcripts from the reaction mixture that would have
influenced the quantification. Afterwards, the RNA was ethanol precipitated and quantified using a
spectrophotometer at 260 nm. Unmodified RNA was synthesized using T7 RNA polymerase wildtype
and mutant M2 in the presence of NTPs. 2’-SeCH3-modified RNA was synthesized using T7 RNA
polymerase mutant M2 in the presence of 2’-SeCH3-UTP. From a 400 µL transcription reaction it was
possible to recover 90 µg (3.12 nmol) of unmodified RNA synthesized by the wildtype T7 RNA
polymerase, 35.3 µg (1.22 nmol) of unmodified RNA and 19.5 µg (0.65 nmol) of 2’-SeCH3-modified
RNA synthesized by the mutant T7 RNA polymerase.
3.1.7
Development of a fluorescence readout based high-throughput screening assay
for the identification of active T7 RNA polymerase variants from mutant
libraries
Towards the directed evolution of T7 RNA polymerase mutants that can efficiently synthesize
2’-SeCH3-modified RNA, a screening platform was established to screen T7 RNA polymerase mutant
libraries in high-throughput. In order to accomplish high-throughput during the screening process,
protein expression and selection of variants with desired properties has to be performed in small
volumes using multiwell plates. For this purpose, optimal conditions for protein expression of T7
RNA polymerase using E. coli BL21 cells in a culture volume of 1 ml were tested using the codonoptimized gene. The expression was performed in 96-deepwell plates. The expression conditions as
well as the composition of the lysis buffer for efficient cell lysis were optimized to achieve the release
of sufficient amounts of proteins for screening (Figure 3.9).
29
3. Results and Discussion
Figure 3.9: SDS-PAGE gel analysis of the expression of T7 RNA polymerase wildtype (T7 RNAP wt) in 96deepwell plates. The expression was performed in a culture volume of 1 ml and compared to cells containing the
empty vector (pGDR11). M: Marker; t0: sample before induction with 1 mM IPTG; t60: sample 1 h after
induction with 1 mM IPTG; t180: sample 3 h after induction; t300: sample 5 h after induction.
Next, a screening assay for the detection of RNA polymerase activity was developed in 384well plates to enable the screening directly with E. coli lysates containing the overexpressed variants.
The assay is based on in-vitro transcription using a reaction mixture containing a double-stranded
DNA template with T7 promotor sequence, NTPs and transcription buffer with MgCl2 for enzymatic
activity. After incubating the reactions mixture with the lysates at 37°C to initiate RNA synthesis in
the presence of an active variant, the DNA template is digested using DNase I and the reaction stopped
by the addition of a buffered solution containing SYBR Green II, a RNA-specific dye for subsequent
fluorescence readout of synthesized RNA using a microplate reader (Figure 3.10).
Figure 3.10: Schematic depiction of the screening assay developed to screen mutant libraries of T7 RNA
polymerase for activity. Enzyme variants that have RNA polymerase activity (A) are identified with the RNAspecific dye SYBR Green II and fluorescence readout whereas inactive mutants (B) are not able to give a
fluorescence signal.
30
3. Results and Discussion
In order to obtain a signal-to-background (S/B) ratio sufficient to distinguish active and
inactive protein variants, the transcription conditions were optimized by performing reactions with E.
coli lysates containing T7 RNA polymerase wildtype in comparison to reactions performed with E.
coli lysates without any T7 RNA polymerase. First, different dilutions of the E. coli lysates with
dilution buffer were tested and screening assays performed as described above. In doing so, it was
observed that dilution of the lysates was required to increase the S/B ratio by lowering the background
activity deriving from components released from E. coli cells after lysis (Figure 3.11 A). Next, the
effect of the used DNA template on the S/B ratio was tested by comparing reactions performed with a
double-stranded DNA template coding for a 53-mer transcript with reactions performed with a doublestranded DNA template coding for an 89-mer transcript. Reactions performed with the short DNA
template resulted only in a poor S/B ration of about 1.5. For reactions conducted with the longer DNA
template, a reasonable S/B ration of about 10 was obtained (Figure 3.11 B). These observations
revealed the binding of SYBR Green II to RNA to be strongly dependent on the length and sequence
of the synthesized RNA transcript. In performing screening assays without DNase I digest, it was
observed that the digestion step was necessary to increase the S/B ratio from approximately 5 to about
10 (Figure 3.11 B). Finally, template and NTP concentrations were optimized to maximize the S/B
ratio and to minimize costs that would have derived from excessive NTP consumption (Figure 3.11
C).
31
3. Results and Discussion
Figure 3.11: Optimization of the screening assay used to screen T7 RNA polymerase mutant libraries. The bar
charts represent the signal-to-background ratio determined by fluorescence readout using E. coli lysates
containing T7 RNA polymerase wildtype (T7 RNAP wt) and E. coli lysates containing no T7 RNA polymerase
(pGDR11). (A) Effect of the dilution of E. coli lysates with different volumes of dilution buffer on the signal-tobackground ratio. (B) Effect of the DNase I digestion step and of the transcripts length on the signal-tobackground ratio. (C) Effect of the used NTP and DNA template concentrations on the signal-to-background
ratio. Error values are indicated as mean (standard deviation).
3.1.8
Construction of a T7 RNA polymerase mutant library by random mutagenesis of
amino acid positions Y639 and H784
Towards the directed evolution of T7 RNA polymerase mutants that can efficiently synthesize
2’-SeCH3-modified RNA by accepting 2’-SeCH3-NTPs as substrates, a mutant library was constructed
by saturation mutagenesis of amino acid positions Y639 and H784. Both amino acid residues are
located in the active site of the polymerase and have been demonstrated to be involved in ribose
recognition events and nucleotide discrimination during catalysis. Thus, the effect of any possible
amino acid substitution at these two positions on the polymerization behaviour of 2’-SeCH3-NTPs
should be investigated. The library was constructed by overlap extension PCR using oligonucleotide
primers where the codons encoding amino acid positions Y639 and H784 were randomized during
oligonucleotide synthesis. These degenerated primers possess a NNK substitution at the respective
32
3. Results and Discussion
codon, whereas N stands for all four nucleobases and K for G and T. The restriction on two
nucleobases at the third codon position results in a reduction of stop codon formation and is therefore
required to avoid premature protein release of inactive protein variants from the ribosome during
protein expression. The randomized oligonucleotide primers were used in PCR reactions to produce
DNA fragments containing each of the randomized regions. The PCR products were purified and
assembled in a second step by assembly PCR. Afterwards, the assembled PCR products were cloned
into pGDR11-NdeI using NdeI and HindIII restriction sites, a vector in which a second on-vector NdeI
restriction site was removed by introducing a silent mutation. Electrocompetent E. coli BL21 cells
were transformed with the resulting library and cultivated on LB-agar plates supplemented with the
respective selection marker overnight. A total number of 3200 single colonies was picked to cover
95% of the relevant protein sequence space due to oversampling and cultivated into 384-deepwell
plates for long term storage at -80°C. E. coli cells containing plasmids encoding the wildtype T7 RNA
polymerase and cells containing the empty pGDR11 vector were placed as controls on all 384deepwell stock plates. Codon randomization of amino acid positions Y639 and H784 was verified by
DNA sequencing of 10 selected transformants. Thereby, it was observed that all 10 selected clones
possessed different amino acid substitutions at both of the randomized positions (Table 3.1).
Table 3.1: Results of the DNA sequencing of 10 selected cloned from the mutant library in which amino acid
positions Y639 and H784 were randomized.
3.1.9
Expression and screening of the mutant library in which amino acid positions
Y639 and H784 were randomized for active variants
Mutants from the T7 RNA polymerase library were expressed in 96-deepwell plates in a
culture volume of 1 mL for 5 h at 37°C. Cells were harvested by centrifugation and lysed by the
addition of lysozyme containing lysis buffer. Afterwards, lysates were diluted by the addition of a
33
3. Results and Discussion
fourfold volume of dilution buffer and cleared by centrifugation. The supernatants containing the
overexpressed T7 RNA polymerase mutants were directly used for activity screening (Figure 3.12).
Figure 3.12: SDS-PAGE gel analysis of supernatants containing overexpressed T7 RNA polymerase variants.
M: Marker; 1, 2: Positive control (T7 RNA polymerase wildtype); 3, 4: Negative control (pGDR11); 5-14: T7
RNA polymerase variants from the library.
For this purpose, the supernatants were transferred into 384-well assay plates containing the
reaction mixture for in vitro transcription by use of a pipetting robot for fast and accurate processing.
The reaction mixture was composed of a double-stranded DNA template with T7 promotor sequence,
NTPs, and transcription buffer containing Mg2+-ions for enzyme activity. The assay plates were
incubated at 37 °C for 90 min to initiate RNA synthesis in the presence of active mutants. Afterwards,
DNA templates were digested using DNase I and the assay stopped by the addition of a buffered
solution containing EDTA and SYBR Green II, a RNA specific stain for subsequent fluorescence
readout of synthesized RNA using a multiwell plate reader with an excitation wavelength of 485 nm
and an emission wavelength of 520 nm. The digestion of the DNA template by DNase I was necessary
to increase the signal-to-background ratio because SYBR-Green II also binds to double-stranded DNA
and would thus interfere with the measurement of synthesized RNA. To distinguish between active
and inactive variants, T7 RNA polymerase wildtype was used as positive control and empty pGDR11
plasmid as negative control. Therefore, E. coli cells containing the T7 RNAP wildtype-pGDR11
plasmid or the empty pGDR11 plasmid were present on all 96-deepwell plates and were processed
along with the mutants to guarantee identical expression and cell lysis conditions. A considerable
signal-to-background ratio of about 10 was obtained. Screening of the 3200 mutants from the library
resulted in 55 active variants that retained at least 30 % activity related to the T7 RNA polymerase
wildtype positive control as determined by fluorescence readout (Figure 3.13).
34
3. Results and Discussion
Figure 3.13: Fluorescence readings from a representative 384-well screening plate of the library where amino
acid positions Y639 and H784 were randomized. Fluorescence intensities were determined by fluorescence
readout using a microplate reader with excitation at 485 nm and emission at 520 nm. (A) Represented values
show arbitrary fluorescence units of a 384-well plate that were calculated by subtracting the mean fluorescence
of the assay blank (pGDR11) from all readings. Positive controls (T7 RNA polymerase wildtype) were present in
columns 1, 7, 13 and 19 in rows A-H. Negative controls (pGDR11) were present in columns 1, 7, 13 and 19 in
rows I-P. 320 variants were present in the remaining wells of the 384-well plate. Positive hits that showed at
least 30% activity relative to the mean value of the wildtype positive controls were selected and are highlighted
in gray. (B) Bar chart depicting the results from the representative 384-well plate. Variants labeled with an
asterisk (*) showed at least 30% activity relative to the mean value of the wildtype positive controls and were
selected.
3.1.10 Purification and characterization of identified hits regarding the incorporation of
2’-modified NTPs
The characterization of the identified hits concerning their activity and their acceptance of 2’modified NTPs was investigated by using the purified enzymes in
32
P-based in vitro transcription
35
3. Results and Discussion
reactions. Therefore, the 55 hits were expressed in E. coli BL21 cells and the T7 RNA polymerase
mutants purified via the N-terminal 6×His-tag by use of 96-well filter plates filled with Ni-IDA
chelating sepharose. The purified mutants were adjusted to the same concentration and tested for
activities in the presence of natural NTPs. Reaction products were analyzed by denaturing PAGE and
phosphorimaging. In doing so, the activity of all 55 tested mutants on the provided DNA template with
NTPs was confirmed validating that our screening system is suitable to screen mutant libraries of T7
RNA polymerase for active variants.
Next, the mutants were tested for an increased acceptance of 2’-O-methyl-2’-deoxyuridine-5’triphosphate (2’-OCH3-UTP). The 2’-OCH3-modification is isosteric to the 2’-SeCH3-modification.
Compared to the chemical synthesis of 2’-SeCH3-UTP, the synthesis of 2’-OCH3-UTP starting from
the commercially available nucleoside is less work-intense. 2’-OCH3-UTP was synthesized starting
from nucleoside according to a protocol reported by Kovács and Ötvös.193 The 2’-OCH3-modified
nucleoside was first phosphorylated using POCl3 and then in situ reacted to the 5’-triphosphate by the
addition of pyrophosphate in the presence of 1,8-bis(dimethylamino-naphthalene). The synthesis
yielded in high amounts of 2’-OCH3-UTP for initial incorporation assays. Transcription reactions
including the synthesized 2’-OCH3-UTP followed by denaturing PAGE gel analysis revealed 10
variants with increased incorporation efficiencies of the 2’-OCH3-modification into RNA. These
variants have further been tested by in vitro transcription for an increased acceptance of 2’-SeCH3UTP in comparison to reactions performed with T7 RNA polymerase wildtype and mutant M2,
respectively. To guarantee comparability, the concentrations of all tested T7 RNA polymerases were
adjusted to the same level (Figure 3.14).
Figure 3.14: SDS-PAGE gel analysis showing T7 RNA polymerase variants selected from the mutant library.
Concentrations were adjusted to the same level (2 µM). M: Marker; 1-10: Selected T7 RNA polymerase
variants; 11: T7 RNA polymerase wildtype; 12: T7 RNA polymerase mutant M1; 13: T7 RNA polymerase
mutant M2 .
36
3. Results and Discussion
Denaturing PAGE gel analysis demonstrated that the 10 mutants were also able to incorporate
2’-SeCH3-nucleotides into RNA. The genes of the 10 selected mutants were sequenced and analyzed
revealing that these hits were composed of only three different variants. Among these variants, the
double mutant Y639F, H784A, the mutant Y639F and the mutant Y639M were identified. PAGE gel
analysis indicated that the 3 mutants can incorporate the 2’-OCH3- and 2’-SeCH3-modifications into
an 89-mer transcript, but often stalled at positions when uridine had to be incorporated resulting in a
mixture of truncated transcripts (Figure 3.15, lane 3 and 4). Thus, less full-length products were
formed in comparison to reactions performed with mutant T7 RNA polymerase mutant M2 that is able
to synthesize modified full-length transcripts without stalling (Figure 3.15, T7 RNAP M2, lane 3 and
4). The wildtype T7 RNA polymerase could not incorporate the 2’-SeCH3-modification into RNA,
however, small amounts of full-length transcripts and truncated transcripts were formed in reactions
with 2’-OCH3-UTP (Figure 3.15, T7 RNAP wt, lane 3).
Figure 3.15: Transcription assays with 2’-modified UTP using selected mutants from the library of T7 RNA
polymerase in which amino acid positions Y639 and H784 were randomized. Activity of the mutants was
compared to reactions with T7 RNA polymerase wildtype (T7 RNAP wt) and mutant M2 (T7 RNAP M2).
Transcription of an 89 nt long RNA sequence was performed for 2 h at 37 °C. Transcripts were labeled by the
inclusion of [α32P]GTP and analyzed by denaturing PAGE (10%). Lane 1: Transcription in the presence of ATP,
CTP, GTP, and UTP (+UTP); Lane 2: Transcription in the presence of ATP, CTP, and GTP, but no UTP (-UTP);
Lane 3: Transcription in the presence of ATP, CTP, GTP, and 2’-OCH3-UTP (+2’-OCH3-UTP) ; Lane 4:
Transcription in the presence of ATP, CTP, GTP, and 2’-SeCH3-UTP (+2’-SeCH3-UTP). Reactions with all four
NTPs (lane 1) were diluted tenfold prior to loading for ease of quantification.
37
3. Results and Discussion
3.1.11 Construction of a randomized T7 RNA polymerase mutant library based on
mutant M2 (E593G, Y639V, V685A, H784G) by error-prone PCR
A second T7 RNA polymerase mutant library was constructed by random mutagenesis of
mutant M2 (E593G, Y639V, V685A, H784G). By randomizing the gene of this mutant, nonpredictable amino acid positions that might effect processing of 2’-modified nucleotides should be
identified. For this purpose, the gene of this mutant was amplified by error-prone PCR, a method to
introduce random mutations into a PCR product by Taq DNA polymerase which is inaccurate in the
presence of high concentrations of Mg2+-ions, an imbalanced dNTPs mixture and Mn2+-ions.100 The
determination of an adequate Mn2+-ion concentration used in the PCR reaction is crucial because an
excessive level of mutagenesis would result in a low quality library with a high number of inactive
polymerase variants. For this purpose, PCR reactions were performed in the presence of an increasing
amount of Mn2+-ions ranging from 0.05 – 4 mM. Afterwards, the PCR products were visualized on an
agarose gel demonstrating that the amount of PCR product decreased in the presence of higher
manganese concentrations (Figure 3.16).
Figure 3.16: Agarose gel showing PCR products amplified under error-prone conditions in the presence of an
increasing concentration of Mn2+-ions.
Error-prone PCR was conducted in the presence of 0.1 mM and 0.3 mM Mn2+-ions and the
quality of the resulting libraries checked after cloning the respective PCR products into the pGDR11
vector using SphI and HindIII restriction sites. E. coli BL21 cells were transformed with the plasmids
containing the randomized genes and single colonies grown overnight. From each library, several
single colonies were picked and colony PCR reactions performed. The PCR products were visualized
after agarose gel electrophoresis. PCR products were observed in 6 out of 7 reactions for the library
constructed with 0.1 mM MnCl2 and in 2 out of 8 reactions for the library constructed with 0.3 mM
MnCl2. For this reason, the T7 RNA polymerase mutant library was constructed using a manganese
concentration of 0.1 mM during error-prone PCR. After cloning the randomized PCR products into the
38
3. Results and Discussion
pGDR11 vector, E. coli BL21 cells were transformed with the library. Single colonies were grown
overnight and 1600 transformants picked and cultivated into 384-deepwell plates for long term storage
at -80°C.
3.1.12 Expression and screening of the randomized mutant library for active variants
Expression and screening of the randomized T7 RNA polymerase mutant library was
conducted as described before (3.1.9). To distinguish between active and inactive variants, T7 RNA
polymerase mutant M2 (E593G, Y639V, V685A, H784G) was used as positive control and empty
pGDR11 vector as negative control. E. coli cells containing the respective plasmids were present on
all 384- and 96-deepwell plates and were processed along with the mutants to guarantee homogeneity
during expression and cell lysis. Fluorescence measurements revealed a signal-to-background ratio
(S/B) of about 2. This significant lower S/B ratio in comparison to the screening in which the wildtype
T7 RNA polymerase was used as positive control derived from the lower activity of mutant M2 in
reactions in which natural NTPs are assayed. Cells containing the T7 RNA polymerase wildtype
plasmid were also present on all 96-deepwell and 384-well assay plates to have an overall positive
control for the screening procedure. Screening of the 1600 mutants from the library yielded in 38
active variants that showed at least the same level of activity as the parental mutant M2 as determined
by fluorescence readout (Figure 3.17).
39
3. Results and Discussion
Figure 3.17: Fluorescence readings from a representative 384-well plate resulting from the screening of the
second mutant library that was constructed by randomizing the gene of mutant M2 (E593G, Y639V, V685A,
H784G) by error-prone PCR. Fluorescence intensities were determined by fluorescence readout using a
microplate reader with excitation at 485 nm and emission at 520 nm. (A) Represented values show arbitrary
fluorescence units of a 384-well plate that were calculated by subtracting the mean fluorescence of the assay
blank (pGDR11) from all readings. Controls with T7 RNA polymerase wildtype were present in columns 1, 7,
13 and 19 in rows A-D. Positive controls (mutant M2) were present in columns 1, 7, 13 and 19 in rows E-L.
Negative controls (pGDR11) were present in columns 1, 7, 13 and 19 in rows I-P. Positive hits that showed at
least the same level of activity as the mean value of the positive control with parental mutant M2 were selected
and are highlighted in gray. (B) Bar chart depicting the results from the representative 384-well plate. Mutants
labeled with an asterisk (*) showed at least the same level of activity as the parental mutant M2 and were
selected.
40
3. Results and Discussion
3.1.13 Purification and characterization of identified hits regarding the incorporation of
2’-modified NTPs
The 38 hits from the randomized T7 RNA polymerase mutant library were expressed and
purified via the N-terminal 6×His-tag by affinity chromatography using 96-well filter plates and NiIDA sepharose. T7 RNA polymerase wildtype and mutant M2 (E593G, Y639V, V685A, H784G) were
expressed and purified in parallel for control reactions. The purified hits were tested for activity with
natural NTPs and acceptance of 2’-SeCH3-UTP by 32P-based in vitro transcription. Reaction products
were analyzed by denaturing PAGE and phosphorimaging. In doing so, only the activity of four hits
could be confirmed. These four hits possessed also increased incorporation efficiencies of the 2’SeCH3-modification into RNA in reactions with 2’-SeCH3-UTP replacing UTP in the presence of
ATP, CTP, and GTP compared to reactions performed using the wildtype T7 RNA polymerase. For
the other selected hits it was not possible to detect any polymerase activity. SDS-PAGE gel analysis of
protein expression and of the different purification steps of several of those hits revealed that no
proteins with the correct molecular weights were expressed and that it was not possible to purify them
in any detectable amounts. DNA sequencing of these variants demonstrated that the respective genes
contained different numbers of mutations and also contained a mutation, a deletion or an insertion
leading to an internal stop codon. This internal stop codon resulted in premature protein release from
the ribosome during expression and thereby resulted in truncated proteins without any RNA
polymerase activity. The genes of the four active hits were also sequenced and analyzed. Thereby, it
was discovered that one of those hits had no mutation at all and that two other hits contained three or
four mutations, but silent mutations resulting in no amino acid substitution on protein level. Thus,
three of the hits were identified as the parental mutant M2 (E593G, Y639V, V685A, H784G). The
remaining hit was identified as a mutant containing seven additional mutations along with the four
initial mutations. This mutant was referred to as 2P16 (I119V, G225S, K333N, D366N, F400L,
S661G, F880Y, E593G, Y639V, V685A, H784G). The mutant 2P16, T7 RNA polymerase wildtype
and T7 RNA polymerase mutant M2 were again expressed in E. coli and purified in large scale for
characterization. The incorporation efficiencies of 2’-SeCH3- and 2’-OCH3-modified nucleotides of
mutant 2P16 were compared to those obtained with the parental mutant M2. The mutants were
therefore adjusted to the same protein concentration as verified by SDS-PAGE gel analysis (Figure
3.18).
41
3. Results and Discussion
Figure 3.18: SDS-PAGE gel analysis of purified T7 RNA polymerase mutant 2P16 (2P16) and T7 RNA
polymerase mutant M2 (E593G, Y639V, V685A, H784G). Proteins were adjusted to the same concentration. M:
Marker.
In vitro transcription assays in which UTP was replaced by 2’-SeCH3-UTP or 2’-OCH3-UTP
in the presence of ATP, CTP, and GTP were performed. Furthermore, reactions with NTPs were
conducted as positive controls and reactions without UTP but with ATP, CTP and GTP as negative
controls (Figure 3.19, lane 1 and 2). Denaturing PAGE gel analysis indicated that mutant 2P16
exhibits incorporation efficiencies comparable to those obtained with mutant M2 in reactions with
NTPs and in reactions with 2’-OCH3-UTP, ATP, CTP, and GTP (Figure 3.19, lane 1 and 3). When
only ATP, CTP and GTP were added, no product formation was observed (Figure 3.19, lane 2). In
reactions with 2’-SeCH3-UTP, ATP, CTP, and GTP, increased incorporation efficiencies of the 2’SeCH3-modified nucleotides into the 89-mer transcript were detected for mutant 2P16 (Figure 3.19,
lane 4).
42
3. Results and Discussion
Figure 3.19: Transcription assays with 2’-modified UTP comparing T7 RNA polymerase mutant 2P16 with the
parental mutant M2 (E593G, Y639V, V685A, H784G). Transcription of an 89 nt RNA sequence was performed
for 1 h at 37 °C. Transcripts were labeled by the inclusion of [α32P]GTP and analyzed by denaturing PAGE
(10%). Lane 1: Transcription in the presence of ATP, CTP, GTP, and UTP (+UTP); Lane 2: Transcription in the
presence of ATP, CTP, and GTP, but no UTP (-UTP); Lane 3: Transcription in the presence of ATP, CTP, GTP,
and 2’-OCH3-UTP (+2’-OCH3-UTP); Lane 4: Transcription in the presence of ATP, CTP, GTP, and 2’-SeCH3UTP (+2’-SeCH3-UTP).
These results were confirmed by a time-course experiment. For this purpose, in vitro
transcription assays were performed and samples withdrawn from the reaction mixtures at different
time points between 5 min and 2 h and immediately stopped by the addition of an equal volume of
stop solution containing 50 mM EDTA.. Comparing total intensities of the synthesized full-length
products after 2 h of transcription revealed mutant 2P16 to be able to synthesize approximately twice
as much of the 2’-SeCH3-modified RNA than the parental mutant M2 (Figure 3.20).
43
3. Results and Discussion
Figure 3.20: Time-course experiment for the enzymatic synthesis of 2’-SeCH3-modified RNA using T7 RNA
polymerase mutant 2P16 and mutant M2. Aliquots of the reaction mixtures were withdrawn at the corresponding
time points and quenched with an equal volume of stop solution containing 50 mM EDTA.
In order to gain further insights into the substrate spectra of the identified T7 RNA polymerase
mutant 2P16, its ability to accept a variety of 2’-modified CTPs as substrates was investigated. For this
purpose,
32
P-based in vitro transcription reactions were conducted by replacing CTP with 2’-OCH3-
CTP, 2’-SeCH3-CTP, 2’-NH2 or 2’-F-CTP, respectively, in the presence of ATP, UTP, and GTP. The
used DNA template coded for an 89-mer RNA transcript in which the RNA polymerases had to
incorporate 12 cytidine moieties. PAGE gel analysis revealed that mutant 2P16 is able to incorporate
the different 2’-modified cytidine nucleotides into RNA with at least the same efficiencies as observed
for the parental mutant M2 (Figure 3.21, lane 3-6). In reactions with 2’-OCH3- and 2’-SeCH3-CTP,
T7 RNA polymerase mutant 2P16 exhibited even slightly increased incorporation efficiencies that T7
RNA polymerase mutant M2. The best incorporation efficiencies were obtained with 2’-F- and 2’OCH3-CTP, whereas 2’-SeCH3- and 2’-NH2-CTP were only poor substrates for both T7 RNA
polymerase mutants (Figure 3.21).
44
3. Results and Discussion
Figure 3.21: Transcription assays with 2’-modified CTPs comparing T7 RNA polymerase mutant 2P16 with the
parental mutant M2 (E593G, Y639V, V685A, H784G). Transcription of an 89 nt RNA sequence was performed
for 1 h at 37 °C. Transcripts were labeled by the inclusion of [α32P]GTP and analyzed by denaturing PAGE
(10%). Lane 1: Transcription in the presence of ATP, UTP, GTP, and CTP (+CTP); Lane 2: Transcription in the
presence of ATP, UTP, and GTP, but no CTP (-CTP); Lane 3: Transcription in the presence of ATP, UTP, GTP,
and 2’-OCH3-CTP (+2’-OCH3-CTP); Lane 4: Transcription in the presence of ATP, UTP, GTP, and 2’-SeCH3CTP (+2’-SeCH3-CTP); Lane 5: Transcription in the presence of ATP, UTP, GTP, and 2’-F-CTP (+2’-F-CTP);
Lane 6: Transcription in the presence of ATP, UTP, GTP, and 2’-NH2-CTP (+2’-NH2-CTP).
3.1.14 Discussion of the results and conclusion
By performing in vitro transcription reactions using distinct mutants of T7 RNA polymerase
with increased tolerance towards 2’-modified nucleotides, the enzymatic synthesis of 2’-SeCH3-RNA
was investigated. For this purpose, a variety of interesting RNA target sequences with known
crystallisation behaviour were chosen that should represent the proof-of-principle targets. The first
RNA sequence tested to be synthesized in the presence of either NTPs or in the presence of 2’-SeCH3UTP, ATP, CTP, and GTP was a 27 nucleotides mimic of the E. coli 23S rRNA sarcin/ricin loop
(SRL).190 The T7 RNA polymerase mutants M1 (Y639F, H784A) and M2 (E593G, Y639V, V685A,
H784G) were tested as well as the wildtype polymerase. It was observed that all RNA polymerases
had significant problems with the synthesis of this RNA target sequence. A reasonable amount of full-
45
3. Results and Discussion
length products could only be observed in reactions with T7 RNA polymerase wildtype in the
presence of natural NTPs. No transcription products were formed in reactions containing 2’-SeCH3UTP. In general, transcription can be divided into two phases: initiation and elongation. It is well
known that the initiation-complex is unstable and prone to dissociate until the polymerase proceeds
beyond the first 9-10 residues.88 Additionally, it is also known that transcript yields are greatly
influenced by the composition and sequence of the first three to six nucleotides and that purines,
especially guanosines, are preferred at this positions.65 The tested SRL-sequence (5’-UGCUCCUA
GUACGAGAGGACCGGAGUG-3’) does not contain a purine-rich 5’-initiation site and contains four
uridines until the critical number of about 10 nucleotides required for efficient transcription is reached.
In reactions in which UTP was replaced with 2’-SeCH3-UTP, the polymerase was forced to
incorporate the 2’-methylseleno-modification into the transcript during the initiation phase. Therefore,
the SRL-sequence represented a difficult target sequence for T7 RNA polymerase. After these initial
experiments, the transcription of two more RNA sequences mimicking the E. coli sarcin/ricin loop was
investigated. Both sequences included the tested 27 nucleotides sequence, but were elongated at both
ends for either three (33-mer) or four (35-mer) adjacent nucleotides, thus resulting in a guanosine
containing initiation site. Reactions were only performed in the presence of natural NTPs to
investigate whether those sequences were generally suited for enzymatic synthesis using T7 RNA
polymerase. Even though in vitro transcription of these modified SRL-sequences was observed to be
much more efficient compared to the previously tested 27-mer SLR sequence when using T7 RNA
polymerase wildtype, no transcription products could be observed when using the T7 RNA
polymerase mutants under the same conditions. The same observation was made for the transcription
of another 27 nucleotides sequence, the HIV-1 TAR RNA element that also starts on two
guanosines.191 This might suggest that efficient transcription using both T7 RNA polymerase mutants,
the double mutant M1 (Y639F, H784A) and the quadruple mutant M2 (E593G, Y639V, V685A,
H784G), is even more dependent on the transcript sequence and length than in vitro transcription using
T7 RNA polymerase is in general. Thus, it was assumed that the mutants might have problems with
the short synthetic DNA oligonucleotides that were used as templates and that have been annealed
with the T7 promotor sequence to form a double-stranded promoter region prior to the transcription
reaction. For this reason, the T7 RNA polymerase mutants and the wildtype polymerase were tested
using a double-stranded DNA template containing the T7 promotor sequence that was prepared by
PCR. The template coded for a 29 nucleotides long RNA fragment deriving from helix 6 of the human
signal recognition particle (hSRP) attached to a hammerhead ribozyme at the 3’-end which is cleaved
off from the target sequence co-transcriptionally, thus producing a homogeneous 3’-end. The same
construct was successfully used by Wild et al. for the preparation of 5-bromo-modified RNA using T7
RNA polymerase wildtype and for the structure determination of the hSRP RNA by multiple
anomalous dispertion (MAD).192 The use of such hammerhead ribozyme minimal motifs incorporated
at the 5’-and/or 3’-end of transcription product is a widely used method to avoid 3’-heterogeneity of in
46
3. Results and Discussion
vitro synthesized RNA and to increase transcription efficiency by introducing a purine-rich initiation
site at the 5’-end that will be afterwards cleaved off.194 Indeed T7 RNA polymerase wildtype was
observed to be efficiently synthesizing the unmodified full-length transcripts. The cis-acting ribozyme
was also observed to be efficiently cleaved off from the target sequence resulting in the desired 29mer. However, using this double-stranded DNA template did not increase the amount of unmodified or
2’-SeCH3-modified transcripts synthesized by the T7 RNA polymerase mutants M1 and M2 compared
to reactions performed with the wildtype polymerase.
All efforts to synthesize 2’-SeCH3-modified RNA sequences of high interest failed due to low
transcription efficiencies when using the T7 RNA polymerase mutants M1 and M2. Finally, the
successful enzymatic synthesis of 2’-SeCH3-modified RNA was accomplished by use of a doublestranded DNA template coding for an 89 nucleotides transcript that contained a purine-rich 5’GGGAGA initiation site. Additionally, the template was designed to code for the incorporation of the
first 2’-SeCH3-modified nucleotide (U) at position +10 at which the processive elongation
conformation of the RNA polymerase is achieved. The best incorporation efficiencies of the 2’SeCH3-modification were obtained with T7 RNA polymerase mutant M2 (E593G, Y639V, V685A,
H784G) in reactions with 2’-SeCH3-UTP. T7 RNA polymerase mutant M1 (Y639F, H784A) was also
able to incorporate the 2’-SeCH3-modification into RNA whereas T7 RNA polymerase wildtype fails
to do so. This observation was confirmed by a time-course experiment. Reactions performed in the
presence of 2’-SeCH3-CTP showed the same tendencies, however, the incorporation was much less
efficient. Transcription reactions in the presence of 2’-SeCH3-ATP and 2’-SeCH3-GTP did not yield in
any detectable full-length transcripts for any of the tested T7 RNA polymerases. This observation is
consistent with the observations reported by Chelliserrykattil et al. who could not detect any
incorporation of 2’-OCH3-GTP when using mutant M2.2 As efficient transcription using T7 RNA
polymerase starts with a guanosine-rich sequence and as the stable and processive elongation complex
is formed as soon as the polymerase had incorporated the first 9 to 10 nucleotides, it becomes clear
why the introduction of the 2’-SeCH3-modification by using 2’-SeCH3-GTP is an challenging
undertaking.
Within this work, the enzymatic synthesis of 2’-SeCH3-modified RNA using T7 RNA
polymerase mutants was demonstrated. This approach can especially be used for the preparation of
longer selenium-modified RNA sequences which can not be accomplished by standard chemical solidphase synthesis. Thus, a foundation for an alternative derivatization strategy in X-ray crystallography
for the structure determination of larger RNA sequences was provided that might be acknowledged by
the crystallographic community due to the steadily increasing number of novel non-coding RNA
sequences and the high demand for their structural characterization. In order to make use of the
mutants for in vitro transcription of 2’-SeCH3-modified RNA target sequences in high yields for
crystallisation experiments and to render this approach viable in practice, it might be necessary to
screen reaction conditions using various combinations of concentrations of magnesium and manganese
47
3. Results and Discussion
ions in combination with various nucleotide concentrations to investigate their effect upon
transcription yields. Different ratios and combinations of 2’-SeCH3-NTPs might be tested. The
addition of small amounts of natural UTP to reactions including 2’-SeCH3-UTP might result in an
increased amount of full-length transcripts, however, it must be tested whether the resulting
heterogeneous mixture of RNA sequences containing statistically distributed 2’-SeCH3-modifications
is able to crystallize appropriately. In order to circumvent the difficulties that arise from the sequence
dependency of the T7 RNA polymerase mutants M1 and M2, it might be interesting to test the use of
an all-purine leader sequence that will be post-transcriptional cleaved off by the use of a hammerhead
ribozyme motif attached upstream of the RNA target sequence. The use of such a leader sequence was
reported to largely increase the yields of modified transcripts because it extends beyond the point at
which chain elongation is stable at approximately position +10.159
In the second part of this chapter, a high-throughput screening assay for the identification of
active T7 RNA polymerase variants from mutant libraries has been established. The screening is based
on in vitro transcription and subsequent fluorescence readout of synthesized RNA after the addition of
SYBR Green II, a RNA specific stain. A similar screening technology had been reported by Kuhlman
et al.,195 however, omitting the ultracentrifugation step required in the published screen and the costintensive RNase inhibitor, the simplified screening assay established in this work is much better-suited
for screening libraries in high-throughput. Different steps from the evolved screening procedure
including protein expression in 96-deepwell plates, E. coli lysate preparation and fluorescence-readout
were adapted from a screening developed for the directed evolution of DNA polymerases in our
laboratory.122,
125
In order to obtain an adequate signal-to-background ratio to distinguish between
active and inactive variants, a high level of protein expression in a small culture volume of 1 ml using
96 deepwell plates is required. For this reason, the used gene of T7 RNA polymerase was codonoptimized for protein expression in E. coli. To avoid any interference that could have arised from the
use of commonly used T7 RNA polymerase based E. coli expression systems like pET-vectors in
combination with E. coli BL21(DE3) cells, the mutant libraries were cloned into the pGDR11 vector
that is suitable for protein expression in E. coli BL21 cells under control of a IPTG inducible T5
promotor. Protein expression and the screening assay conditions were optimized using E. coli cells
expressing T7 RNA polymerase wildtype as positive control and E. coli cells containing the pGDR11
vector as negative control. Under optimized conditions, fluorescence measurements revealed a low
background activity and showed a considerable signal-to-background ratio of about 10. The use of a
pipetting robot and microplate reader for library processing enabled screening and evaluation of 384
reactions in approximately 3 h. The screening assay was first validated by screening a T7 RNA
polymerase mutant library composed of 3200 variants generated by saturation mutagenesis of amino
acid positions Y639 and H784. 55 active hits were identified that showed at least 30% activity
compared to the T7 RNA polymerase wildtype reaction as determined by fluorescence readout. The
activity of all 55 hits was afterwards confirmed by 32P-based in vitro transcription using the purified
48
3. Results and Discussion
enzymes and natural NTPs. The lack of false positive hits was probably a result of the high signal-tobackground ratio that enabled an unambiguous assignment of positive and negative hits. Among these
active hits, several T7 RNA polymerase variants with increased acceptance of 2’-OCH3-UTP and 2’SeCH3-UTP were identified, but none of them was observed to be more efficient than T7 RNA
polymerase mutant M2 (E593G, Y639V, V685A, H784G). Surprisingly, DNA sequencing revealed
that these hits were composed of only three different variants: the double mutant Y639F/H784A, the
mutant Y639F and the mutant Y639M, all of them already characterized and described in the literature
for an increased tolerance towards 2’-modified NTPs.1,
2, 171
Encouraged by these results, a second
mutant library of T7 RNA polymerase was constructed by random mutagenesis starting from the
quadruple mutant M2 that had been shown to exhibit the best incorporation efficiencies of the tested
2’-SeCH3-NTPs. Thereby, the polymerization behaviour of this mutant regarding 2’-SeCH3-modified
nucleotides should be improved by mutating non-predictable amino acid positions that might increase
processing of 2’-modified nucleotides. To determine the signal-to-background (S/B) ratio for the
screening of this library, E. coli cells expressing the parental T7 RNA polymerase mutant M2 were
used as positive control and E. coli cells containing the pGDR11 vector as negative control. By using
the same expression and assay conditions as for the screening of the first library, it was only possible
to obtain a signal-to-background ratio of about 2. This poor S/B ratio was probably a result of the
lower expression level when expressing the T7 RNA polymerase mutant M2 compared to the
expression level obtained for T7 RNA polymerase wildtype. Unfortunately, any efforts to increase this
poor signal-to-background ratio by testing different expression conditions failed. Screening of the
1600 variants from this library resulted in 38 hits that showed at least the same level of activity than
the parental mutant M2 as determined by fluorescence readout. Characterization of most of those hits
turned out to be impossible as it was not possible to purify them in detectable amounts. By performing
32
P-based transcription assays with natural NTPs, the activity of only four hits could be validated. One
could assume that it was impossible to detect any activity for most of the variants due to their low
expression levels. However, the poor signal-to-background was probably the main reason for this high
number of false positives. Nevertheless, it was possible to identify a T7 RNA polymerase variant that
indeed showed increased incorporation efficiencies of the 2’-SeCH3-modification into an 89
nucleotides transcript in reactions with 2’-SeCH3-UTP compared to the parental mutant M2.
Furthermore, this variant exhibits the same level of activity than the T7 RNA polymerase mutant M2
in reactions with NTPs, 2’-OCH3-UTP, and with other tested 2’-modified nucleotides like 2’-F- and
2’-NH2-CTP, respectively. This mutant was referred to as 2P16 in accordance to the screening plate
and well from which it has derived from. DNA sequencing revealed that this mutant possesses seven
additional mutations along with the initial four mutations. Those 11 mutations were located in the
polymerase’s N-terminal domain (I119V, G225S), thumb domain (K333N, D366N, F400L), finger
domain (E593G, Y639V, S661G, V685A, H784) and palm domain (F880Y), thus distributed over the
entire RNA polymerase (Figure 3.22 A).
49
3. Results and Discussion
Figure 3.22: Location of mutated amino acid positions in the structure of T7 RNA polymerase. (A) Positions of
mutated amino acids found in T7 RNA polymerase mutant 2P16 as seen in the crystal structure of T7 RNA
polymerase in an open-conformation. T7 Lysozym from the original crystal structure is omitted for clarity (PDB
1ARO). (B) Positions of mutated amino acids found in T7 RNA polymerase mutant 2P16 as seen in the crystal
structure of a transcribing T7 RNA polymerase in complex with DNA/RNA (PDB 1H38). Figures were made
using PyMol.
Apart from amino acid positions Y639 and H784, none of the other mutated positions had
been described before to affect the substrate recognition processes of T7 RNA polymerase. However,
amino acid positions F400 seems to be the only one that is directed towards the active site complexing
the DNA/RNA hybrid and might therefore support the transcription of 2’-SeCH3-modified RNA
(Figure 3.22 B). In order to gain further insights into the structural basis for the increased substrate
spectra of T7 RNA polymerase mutant 2P16 and to learn more about the contribution of each
mutation, it might be interesting to crystallize the polymerase in complex with DNA/RNA and an
incoming 2’-SeCH3-UTP. Those crystallization trials are currently under performance in cooperation
with K. Betz from the Welte group (Department of Biology, University of Konstanz). Furthermore,
turning back single amino acid substitutions to their original amino acids by site-directed
mutatagenesis might allow to draw conclusions about the influence of the different mutation. Taken
together, these results demonstrate the benefit of directed protein evolution to identify T7 RNA
polymerase mutants with increased substrate spectra like the ability to accept 2’-modified nucleotides.
Furthermore, the established screening system was shown to be suitable to identify active T7 RNA
polymerase variants from different mutant libraries that could further be tested for altered properties
like the acceptance of modified nucleotides. The identified T7 RNA polymerase mutant 2P16 with
increased acceptance of 2’-SeCH3-NTPs can further be used for the efficient enzymatic synthesis of
2’-SeCH3-modified RNA for application in X-ray crystallography. Additionally, the characterization
of this T7 RNA polymerase variant provides further insights into the complex process of substrate
recognition of 2’-modified nucleotides.
50
3. Results and Discussion
3.2
Studies on the Enzymatic Synthesis of Synthetic Genetic Polymers
3.2.1
Introduction
Synthetic genetic polymers, also referred to as xeno-nucleic acids (XNA), are unnatural
polymers that have been chemically synthesized in order to investigate their structural behaviours,
properties and enzymatic accessibilities. XNAs can be modified at the nucleobases, at the backbone or
at the sugar moiety by chemical synthesis. Among these analogues are the tetrose-based TNA (α-Lthreofuranosyl nucleic acid), LNA (locked nucleic acids; 2’-O-4’-C-methylen-β-D-ribonucleic acids),
HNA (1,4-anhydro hexitol nucleic acids) and CeNA (cyclohexenyl nucleic acids).15, 17-19 However, the
corresponding 5’-triphosphates of most of the unnatural nucleotide analogues are poor polymerase
substrates at full substitution and the polymers poor templates for reverse transcription.196-199 It has to
be considered that without a decoding mechanism XNA synthesis is a ‘dead-end’ because the genetic
information remains locked in XNA.11 The use of engineered polymerases for XNA synthesis, reverse
transcription and replication is a powerful strategy to overcome these drawbacks, but one has to
challenge the stringent substrate specificy of polymerases while maintaining their activity and
fidelity.121 Recently, Holliger and coworkers have evolved DNA polymerase variants by means of
directed evolution and design that support the synthesis and reverse transcription of six synthetic
genetic polymers and thus enable analysis of the information encoded therein.120 These six XNAs in
which the canonical ribofuranose ring of DNA and RNA is replaced by five- or six-membered ring
structures comprised HNAs, CeNAs, TNAs, LNAs, ANAs (arabino nucleic acids) and FANAs (2’fluoro-arabinonucleic acids). This approach is a milestone in this research field and shows that
synthetic genetic polymers are capable of information storage and transfer, two hallmarks of life.
2’-Deoxyxylose nucleic acids (dXNAs) that exhibit an altered 3’-configuration compared to
DNA have been proposed to be a potential candidate for the development of a genetic system
orthogonal to DNA or RNA.200 In general, orthogonal systems do not interfere with other information
systems. An orthogonal nucleic acid should thus be unable to cross pair with DNA or RNA, should be
able to form a stable duplex structure with reasonable chemical and enzymatic stability and should
exhibit a flexible backbone to allow formation of secondary and tertiary structures needed for
information storage and transfer.13,
14
dXNAs are indeed able to self-pair with at least similar
stabilities than DNA duplexes and can not cross-hybridize to DNA or RNA.201,
202
Additionally,
dXNAs have also been shown to be resistant to cellular nucleases.203 Research in the field of dXNAs
and locked dXNAs was started by Seela et al. and Wengel et al..204-206
In the series of small steps and developments towards an orthogonal information system, the
capability of a variety of DNA polymerases of eukaryotic, prokaryotic, and archaic origin to
synthesize dXNA by use of 2’-deoxyxyloadenosine and deoxyxylothymidine-5’-triphosphates
51
3. Results and Discussion
(dXATP, XTTP) was investigated. Thereby, a polymerase should be identified that shows potential to
be evolved into a DNA-dependent dXNA polymerase and for use in an orthogonal information system
into a dXNA-dependent dXNA polymerase. Additionally, the enzymatic synthesis of HNA, ANA and
CeNa by the same set of natural and mutant DNA polymerases and the respective sugar-modified
thymidine triphosphates (hTTP, araTTP, ceTTP) should be investigated. As already reported for the
enzymatic incorporation of TNA-, CeNA- and HNA-nucleotides, DNA polymerases from the Bfamily such as Therminator and Vent (exo-) showed a capacity for the synthesis of these XNAs. Up to
80 consecutively incorporations of TNA nucleotides by Therminator DNA polymerase have been
reported,207 but only a few consecutively incoporations of CeNA and HNA nucleotides (hATP, ceATP,
ceGTP) were achieved.196,
198
For this reason, the dXNA synthesis by different B-family DNA
polymerases and mutants should be investigated. Those mutants included the Therminator DNA
polymerase mutant L408Q, a mutant with increased substrate spectra generated by directed
evolution,114 as well as two novel KOD exo- DNA polymerase mutants (L408Q and L408Q, A485L)
that have been generated by site-directed mutagenesis in analogy to the Therminator L408Q mutant.
Both mutations L408Q and A485L have been demonstrated to increase the substrate tolerance of DNA
polymerases from the B-family in regard to their acceptance of modified nucleoside triphosphates.
3.2.2
Expression and purification of KOD exo- DNA polymerase, mutant L408Q and
mutant L408Q, A485L
The gene of KOD exo- DNA polymerase, the 3’-5’ exonuclease deficient variant (D141A,
E143A) of KOD DNA polymerase, was ordered from Geneart. The nucleotide sequence was
optimized for protein expression in E. coli and the sequence was designed to contain a C-terminal
TEV protease cleavage site for later on removal of the vector coded affinity tag from the protein. The
gene was cloned into the expression vector pET24a using NdeI and HindIII restriction sites by
Geneart. The pET24a vector is suitable for protein expression under control of an IPTG inducible T7
promotor in E. coli BL21 (DE3) cells and provides the target protein with an C-terminal polyhistidine
tag (6×His-tag) for protein purification using immobilized metal ion affinity chromatography. The
KOD (exo-) mutant L408Q and the double mutant L408Q, A485L were generated by site-directed
mutagenesis and multi site-directed mutagenesis starting from the KOD (exo-)-pET24a construct.
Sequences were confirmed by DNA sequencing. Protein expression of KOD (exo-) DNA polymerases
was conducted using E. coli BL21-Gold (DE3) cells. Cells were cultivated in LB-medium
supplemented with the selection marker kanamycin and proteins expressed for 2.5 h at 37°C after the
induction with IPTG. SDS-PAGE gel analysis indicated the expression of KOD exo- DNA
polymerases which possess a molecular weight of about 93 kDa (Figure 3.23 A). For protein
purification, E. coli lysates containing the overexpressed thermostable DNA polymerases were
incubated for 10 min at 85°C to denature E. coli host proteins. After the removal of denatured E. coli
52
3. Results and Discussion
proteins by centrifugation, the KOD exo- DNA polymerases containing a C-terminal 6×His-tag were
purified by immobilized metal ion affinity chromatography using Ni-IDA chelating sepharose on an
ÄKTA purifier chromatography system. SDS-PAGE gel analysis indicated that the purified proteins
were ≥95% pure after this purification step. Protein concentrations were determined via Bradford
protein assay and adjusted to the same value for further investigations (Figure 3.23 B).
Figure 3.23: Expression and purification of KOD (exo-) DNA polymerase and mutants. (A) SDS-PAGE gel
analysis of the expression and purification of KOD (exo-) DNA polymerases mutant L408Q in E. coli BL21GOLD(DE3) cells. M: Marker; t0: Sample before induction with IPTG; t150: Sample 2.5 h after the induction
with IPTG; P: Pellet; FT: Flow-through; S: Supernatant; 4-14: Elution fractions of the nickel affinity
chromatography purification. (B) SDS-PAGE gel analysis of purified KOD (exo-) DNA polymerase (1), KOD
(exo-) DNA polymerase mutant L408Q (2) and KOD (exo-) DNA polymerase mutant L408Q, A485L (3).
Proteins were adjusted to the same concentration.
3.2.3
Incorporation studies of araTTP, hTTP, xyloTTP and ceTTP by various DNA
polymerases and mutants
The action of a variety of sugar-modified thymidine 5’-triphosphates on DNA polymerases
was explored by investigating the ability of different DNA polymerases of eukaryotic, prokaryotic,
and archaic origin from different evolutionary families to accept the modified triphosphates in primer
extension experiments. Two TTP analogues with six-membered ring structures replacing the 2’deoxyribofuranose ring and two analogues with altered 2’- or 3’-OH group configuration were tested.
Those four analogues, arabino-furanosyl thymidine 5’-triphosphate (araTTP), 1,5-anhydrohexitol
thymidine 5’-triphosphate (hTTP), deoxyxylose thymidine 5’-triphosphate (xyloTTP), and
cyclohexenyl thymidine 5’-triphosphate (ceTTP), had been chemically synthesized in the group of
Prof. Dr. Piet Herdewijn from Katholieke Universiteit Leuven in Belgium. Several commonly used
DNA polymerases were tested as well as a variety of DNA polymerase mutants with broadened
substrate spectra. Those mutants had been either generated by site-directed mutagenesis or by directed
53
3. Results and Discussion
protein evolution. All of the used DNA polymerases had been overexpressed and purified in the Marx
group: Klentaq DNA polymerase (KTQ wt), Klentaq double mutant (KTQ DM)208, Taq DNA
polymerase (Taq wt), and the Taq mutant M1 (Taq M1)209 from the A-family and Therminator DNA
polymerase (Therminator wt), Therminator mutant L408Q (Therminator L408Q)114, and 9°N exoDNA polymerase from the B-family. Additionally, human DNA polymerase β (Pol β wt) and the DNA
polymerase β mutant 5P20 (Pol β 5P20)123 from the X-family of DNA polymerases and Dpo4 DNA
polymerase from the error-prone Y-family of TLS polymerases were tested. Primer extension assays
were conducted by using a 23 nucleotides
32
P-labeled primer (BRAF 23C) along with a template
bearing only adenosine residues after the primer binding site coding for 46 incorporations of the
thymidine analogues (69-PolyA (46A)). Thus, it should be investigated how many of the sugarmodified nucleotides can be incorporated consecutively in reactions in which only the sugar-modified
TTP analogues were added, but no other dNTP. Control reactions in the presence of natural TTP and
without any TTP were performed simultaneously. All reactions were afterwards analyzed by
denaturing polyacrylamide gel electrophoresis (PAGE) and phosphorimaging.
In doing so, it was observed that all tested DNA polymerases from the A-family had
significant problems with the sugar-modified TTP analogues, especially Klentaq DNA polymerase for
which an incorporation of only one single anhydrohexitol nucleotide was detected. Reactions with the
Klentaq DNA polymerase double mutant, Taq DNA polymerase and Taq DNA polymerase mutant M1
revealed that the polymerases were able to incorporate one of the modified nucleotides, but had
problems with the extension for another modified nucleotide (Figure 3.24). In contrast, all three tested
DNA polymerases from the B-family were extremely efficient in incorporating the sugar-modified
nucleotides. The best incorporation efficiencies were obtained in reactions with the Therminator DNA
polymerase mutant L408Q that was able to incorporate up to 10 arabinofuranosyl and anhydrohexitol
nucleotides and up to 8 cyclohexenyl nucleotides after the primer strand. In reations with xyloTTP, the
Therminator mutant L408Q was only able to incorporate one nucleotide and to extend it for another
(Figure 3.24). Reactions with the two DNA polymerases Therminator wildtype and 9°N exo- showed
the same tendencies for incorporation, however, both polymerases were not able to reach the high
number of consecutively incorporations that were observed with the mutant L408Q. In reactions
performed with human DNA polymerase β wildtype it was observed that the incorporation of the first
modified nucleotide was quite efficient for all four tested TTP analogues, but the extension for another
nucleotide was inhibited. The DNA polymerase β mutant 5P20 showed improved incorporation
efficiencies of the four modified nucleotides in comparison to the wildtype enzyme and was even able
to elongate the first incorporated nucleotide. As expected, due to the altered configuration of the 3’hydroxyl group of xyloseTTP that is required after incorporation for DNA strand elongation, this TTP
analogue was observed to be the poorest substrate for most of the tested DNA polymerases (Figure
3.24).
54
3. Results and Discussion
Figure 3.24: Primer extension reactions using sugar modified TTP analogues and various DNA polymerases.
Chemical structures of the sugar modified TTP analogues and parts of the used primer template sequences are
depicted above. The employed DNA polymerases are indicated above each set of reactions. P: 32P-labeled
Primer; 1: Control reaction without any dNTP; 2: Control reaction with natural TTP; 3: Reaction in the presence
of araTTP; 4: Reaction in the presence of hTTP; 5: Reaction in the presence of xyloTTP; 6: Reaction in the
presence of ceTTP.
3.2.4
Incorporation studies of 2’-deoxyxylo-ATP and xyloTTP by various DNA
polymerases and mutants
To gain further insights into the enzymatic incorporation of 2’-deoxyxylose nucleotides into a
growing DNA chain, the capability of a variety of DNA polymerases to accept 2’deoxyxyloadenosine-5’-triphosphate (dXATP) and deoxyxylose thymidine 5’-triphosphate (XTTP) as
substrates was investigated in primer extension assays. Previous incorporation experiment revealed the
altered configuration of the 3’-hydroxyl group of 2’-deoxyxylose nucleotides that is required for DNA
strand elongation after incorporation as a significant barrier for the efficient enzymatic synthesis of 2’deoxyxylose nucleic acids. Nevertheless, DNA polymerases from the B-family were quite efficiently
incorporating the other tested sugar-modified nucleotides. For this reason, primer extension reactions
with KOD exo- DNA polymerase, another B-family polymerase with an excellent processivity were
55
3. Results and Discussion
conducted. In analogy to the Therminator DNA polymerase mutant L408Q which is the L408Q,
A485L mutant of 9°N exo- DNA polymerase, KOD exo- DNA polymerase mutant L408Q and the
double mutant L408Q, A485L were generated and tested in primer extension experiments.
Additionally, several commonly used DNA polymerases as well as a variety of DNA polymerase
mutants with broadened substrate spectra were tested. Primer extension assays were conducted by
using a 23 nucleotides 32P-labeled primer (BRAF-23C) along with a template bearing just thymidine
or adenosine residues after the primer binding site coding for 46 incorporations of the respective
sugar-modified analogue (69-PolyA (46A); 69-PolyT (46T)). In doing so, it should be investigated
how many of the 2’-deoxyxylo-modified nucleotides can be incorporated consecutively in reactions in
which only the modified nucleotides were added, but no other dNTP. Control reactions in the presence
of natural dATP/TTP and without any dATP/TTP were performed simultaneously. All reactions were
afterwards analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) and phosphorimaging.
For reactions performed with dXATP, it was observed that all of the tested DNA polymerases
were able to incorporate one 2’-deoxyxylose nucleotide in a template dependent manner, except for
Klentaq DNA polymerase (KTQ wt). Furthermore, most of the DNA polymerases were also able to
extend this first incorporated nucleotide for another 2’-deoxyxylo nucleotide (Figure 3.25). It was also
observed that all of the tested DNA polymerase mutants showed improved extension behaviours
compared to their respective wildtype polymerase. This observation can be exemplified in comparing
reactions performed with KOD exo- DNA polymerase with reactions performed with the two KOD
mutants. Whereas KOD exo- DNA polymerase was only able to extend the primer for one 2’deoxyxylo nucleotide, the KOD DNA polymerase mutant L408Q was able to extend this modified
nucleotide for another one and the KOD exo- DNA polymerase double mutant L408Q, A485L was
even capable of extending the primer more efficiently with up to three modified nucleotides.
Interestingly, human DNA polymerase β mutant 5P20 seems to be most proficient in extending the
primer for up to three 2’-deoxyxyloadenosine moieties whereas Dpo4 DNA polymerase as a member
of the error-prone TLS polymerase Y-family was only moderate efficient in extending the first
incorporated modified nucleotide.
56
3. Results and Discussion
Figure 3.25: Primer extension reactions using xylo-dATP and various DNA polymerases. Chemical structure of
xylo-dATP and parts of the used primer template sequences are depicted above. The employed DNA
polymerases are indicated above each set of reactions. P: 32P-labeled Primer; 1: Control reaction without any
dNTP; 2: Control reaction with natural dATP; 3: Reaction in the presence of xylo-dATP.
For reactions performed with XTTP, similar observations as for reactions performed with
dXATP were made. All of the tested DNA polymerases of eukaryotic, prokaryotic, and archaic origin
were able to incorporate one 2’-deoxyxylo nucleotide except for Klentaq DNA polymerase (KTQ wt)
and most of them were also able to extend this first incorporated nucleotide for another 2’-deoxyxylo
nucleotide (Figure 3.26). All of the tested DNA polymerase mutants showed improved extension
behaviours compared to their respective wildtype polymerase. Unlike as observed in reactions with
xyloATP, the Taq DNA polymerase mutant M1 was able to extend the primer with up to four
xylothymidine moieties. Nevertheless, human DNA polymerase β mutant 5P20 seems again to be
most efficient in extending the primer for up to two 2’-deoxyxylo nucleotides almost quantitatively.
57
3. Results and Discussion
Figure 3.26: Primer extension reactions using xyloTTP and various DNA polymerases. Chemical structure of
xyloTTP and parts of the used primer template sequences are depicted above. The employed DNA polymerases
are indicated above each set of reactions. P: 32P-labeled Primer; 1: Control reaction without any dNTP; 2:
Control reaction with natural TTP; 3: Reaction in the presence of xyloTTP.
3.2.5
Discussion of the results and conclusion
By performing primer extension assays, the capacity of a variety of natural and mutant DNA
polymerases to synthesize the synthetic genetic polymers ANA, CeNA, HNA and dXNA was
investigated. Homopolymer formation was examined by use of a DNA primer-template complex
where the template coded for the consecutively incorporation of 49 sugar-modified nucleotides. In
doing so, a candidate polymerases should be identified that can be used to evolve into DNA-dependent
XNA polymerases. Family B DNA polymerases and mutants efficiently incorporated arabino,
cyclohexenyl and hexitol nucleotides whereas family A DNA polymerases had significant problems
with the sugar-modified nucleotides. These observations had already previously been reported by
Herdewijn and coworkers who studied the enzymatic incorporation of hexitol and cyclohexenyl
nucleotides into DNA by DNA polymerases from both evolutionary families.196,
198
In reactions
performed with Vent (exo-) DNA polymerase in the presence of hATP, the authors observed up to 3
consecutively incorporations and up to six under very harsh conditions whereas only one incorporation
and no elongation was observed in reactions with Taq DNA polymerase. The same tendencies had
been reported for reactions performed with Vent (exo-) and Taq DNA polymerase in the presence of
ceATP. The B-family polymerase was capable to incorporate one modified nucleotides and to extend
this with up to 7 cyclohexenyl nucleotides in the presence of MnCl2 whereas Taq DNA polymerase
was only able to incorporate up to two nucleotides under the same conditions, but with general poor
incorporation efficiencies. In the present work, it was achieved to incorporate 8-10 consecutively
arabino, cyclohexenyl and hexitol nucleotides under standard conditions in the presence of the
58
3. Results and Discussion
respective sugar-modified thymidine triphosphates by using the B-family DNA polymerase mutant
Therminator L408Q. Therminator DNA polymerase mutant L408Q represents a mutant with increased
substrate spectra generated by directed evolution.114 These results indicate that family-B polymerases
might be well-suited to evolve into DNA-dependent XNA polymerases. The general observation that
all tested DNA polymerase mutants with broadened substrate spectra that had either been generated by
site-directed mutagenesis or by directed protein evolution showed increased incorporation behaviours
of the nucleotide analogues compared to their respective wildtype polymerase reveals directed protein
evolution as a powerful approach to engineer efficient XNA polymerases. This suggestion was
supported by the work recently published by Holliger et al., who reported the directed evolution of Bfamily polymerase variants deriving from Tgo, the replicative DNA polymerase of the archaeon
Thermococcus gorgonarious, that are capable of processive XNA synthesis from a DNA template and
that can reverse transcribe XNA back into DNA.120 As a perspective for further investigations it would
be interesting to study the behaviour of the DNA polymerase mutants tested in this work when using
the respective XNA as template, for both reverse transcription into DNA and replication.
The investigations on the DNA-dependent enzymatic synthesis of dXNA have revealed that
xylo-dATP and xyloTTP are poor substrates for all of the tested DNA polymerases, no matter from
which polymerase family they are. This observation is consistent with the proposed orthogonality of
dXNA. Even though incorporation of one xylose nucleotide was achieved by almost all DNA
polymerases, the polymerization was hindered apparently due to the altered configuration of the 3’OH group that is required for stand elongation. However, human DNA polymerase β was surprisingly
efficient in incorporating and extending the xylose nucleotides. Up to 3 incorporations have been
observed in reactions with xylo-dATP even though the main band on the PAGE gel corresponds to 2
incorporations. These results are suggesting DNA-dependent dXNA polymerase activity and might
therefore be an important study on the way towards engineered dXNA polymerases required to copy
existing genetic information from DNA into dXNA. Nevertheless, in order to consider dXNA as
synthetic genetic polymer in an orthogonal system that does not interfere with the consisting DNA and
RNA information systems, it will be necessary to evolve dXNA polymerases that show no activity for
natural substrates as dNTPs and DNA-templates. For this purpose, it might be mandatory to first
investigate the extension of dXNA duplex structures with xylose nucleotides by DNA polymerases to
reveal a starting point for directed evolution.
59
60
4. Materials and Methods
4 Materials and Methods
4.1
Materials
4.1.1
General
Unless otherwise stated, chemicals were used in p.a. or molecular biology grade quality. Water used
for buffers was from a combined reverse osmosis/ultrapure water system (Milli-Q, Sartorius) and
water used for reactions containing RNA and DNA was from a bidistillation apparatus.
4.1.2
Chemicals used for molecular biology
Acetic acid
VWR BDH Prolabo
Acrylamide-bisacrylamide (25%), Rotiphorese
sequencing gel concentrate (19:1)
Roth
Acrylamide-bisacrylamide (30%), Rotiphorese
gel 30 (37.5:1)
Roth
Agar-agar
Roth
Agarose (LE)
Roche
Ammonium sulfate
Roth
Ammonium persulfate (APS)
Roth
Benzamidine hydrochloride hydrate
Roth
Boric acid
Roth
Bromophenol blue
Roth
Calcium chloride
Merck
Carbenicillin disodium salt
Roth
Ni-IDA sepharose (Chelating Sepharose Fast Flow)
GE Healthcare
Chloroform-Isoamyl alcohol (24:1)
Roth
Coomassie Brilliant blue G 250
Roth
Coomassie Roti-Blue (5x)
Roth
Coomassie Roti-Quant (5x)
Roth
Dimethyl sulfoxide (DMSO)
Sigma-Aldrich
1,4-Dithiothreitol (DTT)
Roth
Ethanol (100%)
Sigma-Aldrich
Ethidium bromide (1%)
Roth
61
4. Materials and Methods
Ethylenediaminetetraacetic acid (EDTA) disodium salt
Roth
Formamide
Roth
D(+)-Glucose
Sigma-Aldrich
Glycerol
VWR BDH Prolabo
Glycine
Roth
Hydrochloric acid (37%)
Merck
Imidazole
Merck
Isopropyl-β-D-thiogalactopyranoside (IPTG)
Roth
Kanamycin sulfate
Roth
LB-broth (Lennox)
Roth
Manganese dichloride
Riedel-de Häen
Magnesium chloride hexahydrate
Acros Organics
β-Mercaptoethanol
Merck
N, N, N’, N’-Tetramethylethylenediamine (TEMED)
Roth
Phenol-Chloroform-Isoamyl alcohol (25:24:1)
Roth
Phenylmethylsulfonyl fluoride (PMSF)
Roth
Polyoxyethylene (20) sorbitan monolaurate (Tween 20)
Roth
Potassium chloride
Merck
2-Propanol
Fisher Scientific
Sephadex G-25 Superfine
GE Healthcare
Spermidine
Sigma-Aldrich
SOB-medium (SOB-broth)
Roth
Sodium chloride
VWR BDH Prolabo
Sodium dodecyl sulfate (SDS)
Roth
SYBR Green II RNA gel stain
Fluka
Tris(hydroxymethyl)aminomethane (Trizma Base)
Sigma Aldrich
Triton X-100
Roth
Urea
Roth
Xylene cyanol
Roth
4.1.3
Reagents used for chemical synthesis
Acetic acid
VWR BDH Prolabo
Acetonitrile
VWR BDH Prolabo
1,8-bis(dimethylaminonaphthalene)
Fluka
Dimethylformamide (DMF)
Fluka
62
4. Materials and Methods
Disodium pyrophosphate
Merck
2’-O-methyl-2’-deoxyuridine
Molekula
Tributylamine
Acros Organics
Triethylamine
Merck
Trimethyl phosphate (TMP)
Acros Organics
Phosphoryl chloride
Acros Organics
4.1.4
Nucleotides and radiochemicals
dATP, dCTP, dGTP, TTP
Fermentas, Roche
ATP, CTP, GTP, UTP
Fermentas, Roche
2’-NH2-dCTP, 2’-OCH3-dCTP, 2’-F-CTP
IBA
2’-SeCH3-UTP, -CTP, -ATP, -GTP
T. Santner, Prof. Dr. R. Micura,
Leopold-Franzens University,
Innsbruck, Austria
XTTP, dXATP, araTTP, hTTP, ceTTP
Prof. Dr. P. Herdewijn, Katholieke
Universiteit Leuven, Belgium
[α-32P]GTP (3000 Ci/mmol)
32
[γ- P]ATP (3000 Ci/mmol)
4.1.5
Hartmann Analytic
Hartmann Analytic
Oligonucleotides
Oligonucleotides were obtained from Metabion International AG and Biomers. Cloning primers were
ordered desalted, template oligonucleotides were ordered HPLC purified. Primers used for primer
extension reactions were additionally purified by preparative PAGE and ethanol precipitation. Primers
with randomized positions were purchased HPLC purified from Purimex.
4.1.6
DNA, RNA and Protein Standards
Bovine serum albumin (BSA) standard (2 mg/mL)
Thermo Scientific
Gene Ruler DNA ladder mix
Fermentas
Gene Ruler 1kb DNA ladder
Fermentas
Gene Ruler Low Range DNA Ladder
Fermentas
Low Molecular Weight DNA Ladder
New England Biolabs
63
4. Materials and Methods
Low Range ssRNA Ladder
New England Biolabs
Page Ruler Unstained Protein Ladder
Fermentas
4.1.7
Enzymes and Proteins
Antarctic phosphatase
New England Biolabs
Bovine serum albumin (BSA)
Serva
Calf intestinal alkaline phosphatase (CIAP)
Fermentas
Human DNA polymerase β and mutant 5P20
S. Gieseking,
F. Di Pasquale123
DNase I, RNase-free
Fermentas
DpnI
New England Biolabs
Dpo4 DNA polymerase
F. Streckenbach
HindIII
New England Biolabs, Fermentas
KTQ DNA polymerase and double mutant (DM)
S. Obeid208
Lysozyme
Roth
NdeI
New England Biolabs, Fermentas
9°N DNA polymerase
N. Staiger
Phusion High Fidelity DNA polymerase
Finnzymes
RiboLock RNase Inhibitor
Fermentas
Shrimp alkaline phosphatase (SAP)
Fermentas
SphI (PaeI)
New England Biolabs,
Fermentas
Taq DNA polymerase
Fermentas,
R. Kranaster
Taq DNA polymerase M1
R. Kranaster209
T4 DNA ligase
New England Biolabs
Therminator DNA polymerase and mutant L408Q
N. Staiger114
T4 polynucleotidekinase (PNK)
Fermentas
4.1.8
Kits
DNA Clean & Concentrator-5 Kit
Zymo Research
MinElute Reaction Cleanup Kit
Qiagen
peqGOLD Gel Extraction Kit
PEQLAB
64
4. Materials and Methods
peqGOLD Plasmid Miniprep Kit
PEQLAB
QIAGEN Plasmid Midi Kit
Qiagen
QIAquick Gel Extraction Kit
Qiagen
QIAprep Spin Miniprep Kit
Qiagen
QuikChange Multi Site Directed Mutagenesis Kit
Stratagene
4.1.9
Bacterial strains and plasmids
E. coli XL10-GOLD
Stratagene;
Genotype: Tetr Δ(mcrA)183
Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1
recA1 gyrA96 relA1 lacHte [F¢ proAB lacIqZΔM15
Tn10 (Tetr) Amy Camr]
E. coli BL21-GOLD(DE3)
Stratagene;
Genotype: E. coli B F– ompT hsdS(rB– mB–) dcm+
Tetr gal λ(DE3) endA Hte
E. coli BL21
Amersham Biosciences; AG Boos
Genotype: E. coli B F- ompT hsdS (rB-, mB-) gal
dcm
pGDR11
Ampr, T5 promotor, lac operator, N-terminal
6×His-Tag;
derivative
of
pEQ31
(Qiagen)
q
containing lacI gene
pET24a(+)
Kanr, T7 promotor, lacIq gene, lac operator, Cterminal 6×His-Tag; Novagene
4.1.10 Electrophoresis buffers and solutions
Agarose gel staining solution
0.5x TBE/ 1x TAE supplemented with ethidium
bromide
65
4. Materials and Methods
6x DNA Loading Dye
Fermentas;
10 mM Tris-HCl pH 7.6
0.03% Bromophenol blue
0.03% Xylene cyanol FF
60% Glycerol
60 mM EDTA
Coomassie colloidal staining solution
20% (v/v) Methanol (tech.)
1x Roti-Blue
Coomassie colloidal destaining solution
25% (v/v) Methanol (tech.)
Coomassie methanol
1 L Methanol (tech.)
1 g/L Coomassie Brilliant Blue
G 250; filtrated
Coomassie staining solution
50% (v/v) Coomassie methanol
10% (v/v) Acetic acid (tech.)
Coomassie destaining solution
50% (v/v) Methanol (tech.)
10% (v/v) Acetic acid (tech.)
Denaturing PAGE loading dye
90% (v/v) Formadmide
Transcription assay (stop solution)
50 mM EDTA
0.01% (w/v) Bromophenol blue
0.01% (w/v) Xylene cyanol
Denaturing PAGE loading dye
80% (v/v) formamide
PEX (stop solution)
20 mM EDTA
0.25% (w/v) bromophenol blue
0.25% (w/v) xylene cyanol
Denaturing PAGE gel solution (10%)
10% Acrylamide/ Bisacrylamide (19:1) in
8.3% Urea
1x TBE in 8.3 M Urea
0.08% (w/v) APS
0.04% (v/v) TEMED
66
4. Materials and Methods
Denaturing PAGE running buffer
1x TBE
SDS-PAGE running buffer (10x)
250 mM Tris-HCl pH 8.9
2 M Glycine
1% (w/v) SDS
SDS-PAGE loading dye (6x)
225 mM Tris-HCl pH 6.8
50% (v/v) Glycerol
5% (w/v) SDS
0.05% (w/v) Bromophenol blue
12.5% (v/v) β-Mercaptoethanol
SDS-PAGE separating gel solution (12%)
375 mM Tris-HCl pH 8.8
0.1% (w/v) SDS
12% Acrylamide/ Bisacrylamide (37.5:1)
0.1% (w/v) APS
0.1% (v/v) TEMED
SDS-PAGE stacking gel solution (4%)
250 mM Tris-HCl pH 6.8
0.05% SDS
4% Acrylamide/ Bisacrylamide (37.5:1)
0.1% (w/v) APS
0.25% (v/v) TEMED
TAE-buffer (1x)
40 mM Tris-HCl pH 7.5
40 mM Acetic acid
1 mM EDTA
TBE-buffer (0.5x)
45 mM Trizma Base
45 mM Boric acid
1 mM EDTA pH 8.0
67
4. Materials and Methods
4.1.11 Buffers used in enzymatic reactions
Transcription buffer T7 RNAP (5x)
200 mM Tris-HCl pH 8.0
10 mM Spermidine
50 mM DTT
30 mM MgCl2
KlenTaq reaction buffer (1x)
50 mM Tris-HCl pH 9.2
16 mM (NH4)2SO4
2.5 mM MgCl2
0.1% Tween 20
ThermoPol buffer NEB (1x)
20 mM Tris-HCl pH 8.8
10 mM (NH4)2SO4
10 mM KCl
2 mM MgSO4
0.1% Triton X-100
DNA polymerase β reaction buffer (1x)
50 mM Tris-HCl pH 8.0
20 mM NaCl
20 mM KCl
10 mM MgCl2
2 mM DTT
1% (v/v) Glycerol
200 µg/mL BSA
Dpo4 DNA polymerase reaction buffer (1x)
50 mM Tris-HCl pH 8.0
10 mM MgCl2
20 mM KCl
2 mM DTT
100 µg/mL BSA
10% Glycerol
68
4. Materials and Methods
KOD DNA polymerase reaction buffer (1x)
120 mM Tris-HCl pH 8.0
10 mM KCl
6 mM (NH4)2SO4
0.1% (v/v) Triton X-100
0.001% (w/v) BSA
1.5 mM MgCl2
4.1.12 Buffers and solutions used for screening
Lysis buffer screening T7 RNAP
50 mM Tris-HCl pH 8.0
300 mM NaCl
0.1 % Triton X-100
0.1 mg/mL Lysozyme
Dilution buffer screening T7 RNAP
50 mM Tris-HCl pH 8.0
100 mM NaCl
5 mM DTT
DNase I dilution buffer
50 mM Tris-Acetat pH 7.5
10 mM CaCl2
SYBR Green II stop solution
50 mM Tris-HCl pH 8.0
25 mM EDTA
4x SYBR Green II
4.1.13 Buffers and solutions used for protein purification
Lysis buffer T7 RNAP
50 mM Tris-HCl pH 8.0
300 mM NaCl
0.1 % Triton X-100
0.1 mg/mL Lysozyme
1 mM PMSF
1 mM Benzamidine
69
4. Materials and Methods
Lysis buffer KOD DNA polymerase
20 mM Tris-HCl pH 8.0
300 mM NaCl
0.1% (v/v) Triton X-100
5 mM imidazole
0.1 mg/ml lysozyme
1 mM Benzamidine
1 mM PMSF
IMAC buffer A (T7 RNAP)
50 mM Tris-HCl pH 8.0
300 mM NaCl
0.1 % Triton X-100
IMAC buffer B (T7 RNAP)
50 mM Tris-HCl pH 8.0
300 mM NaCl
0.1 % Triton X-100
250 mM Imidazole
IMAC binding buffer (T7 RNAP)
50 mM Tris-HCl pH 8.0
300 mM NaCl
5 mM Imidazole
0.1 % Triton X-100
IMAC washing buffer (T7 RNAP)
50 mM Tris-HCl pH 8.0
300 mM NaCl
20 mM Imidazole
0.1 % Triton X-100
IMAC buffer A (KOD DNAP)
20 mM Tris-HCl pH 8.0
300 mM NaCl
0.1 % Triton X-100
IMAC buffer B (KOD DNAP)
20 mM Tris-HCl pH 8.0
300 mM NaCl
0.1 % Triton X-100
250 mM Imidazole
70
4. Materials and Methods
Storage buffer T7 RNAP (2x)
100 mM Tris-HCl pH 8.0
200 mM NaCl
10 mM DTT
2 mM EDTA
0.2% (v/v) Triton X-100
Storage buffer T7 RNAP (20x)
100 mM Tris-HCl pH 8.0
200 mM NaCl
100 mM DTT
20 mM EDTA
0.2% (v/v) Triton X-100
Storage buffer KOD DNA polymerase (2x)
100 mM Tris-HCl pH 8.0
100 mM KCl
2 mM DTT
0.2 mM EDTA
0.2% Triton X-100
4.1.14 Media
LB-medium
20 g/L LB-broth (Lennox);
autoclaved
LB-agar plates
20 g/L LB-broth (Lennox)
20 g/L Agar-agar; autoclaved
SOB-medium
20 g/L SOB-broth; autoclaved
SOC-medium
SOB-medium
20 mM Glucose
4.1.15 Antibiotics
Carbenicillin
100 mg/mL
Kanamycin
34 mg/L
71
4. Materials and Methods
4.1.16 Instruments
Agarose gel system
Fisher Biotech
ÄKTA purifier (Unicorn 5.20)
GE Healthcare
Analytical balance (PG403-S, AJ150)
Mettler Toledo
Autoclave (3150 ELV)
Systec
Balance (PE1600)
Mettler Toledo
Block heater
Stuart, Grant Instruments
Centrifuge (5810R)
Eppendorf
Centrifuge (Multifuge 4 KR)
Heraeus
Centrifuge (Biofuge primo R)
Heraeus
Centrifuge (MiniSpin)
Eppendorf
Centrifuge (Rotilabo Uni-fuge)
Roth
Contamat (S.E.A. CoMo170)
Melit
Cooling system pipetting robot
(DL10, K20)
Thermo Haake
Electroporator (GenePulser Xcell)
Biorad
ESI-IT-MS (esquire 3000+)
Bruker Daltonics
ESI-MS (micro-TOF II)
Bruker Daltonics
Freezer -20°C (profi line)
Liebherr
Freezer -80°C (-86 ULT Freezer)
Thermo Scientific Forma
Fridge 4°C (Premium)
Liebherr
Gel analyzer
BIORAD
Gel dryer (Model 583)
BIORAD
Incubator
Memmert
Incubator shaker (Innova 44)
New Brunswick Scientific
Incubator shaker 96-well plates
(Inkubator 1000, Titramax 1000)
Heidolph
Magnetic stirrer (MR 3000)
Heidolph
Microplate reader (POLARstar OPTIMA)
BMG
MPLC system
Büchi
Multidrop (Combi)
Thermo Scientific
Nanodrop Spectrophotometer (ND-1000)
PEQLAB
NMR Avance III 400 MHz spectrometer
Bruker
Overhead shaker (Reax 2)
Heidolph
PAGE system (Sequi Gen GT)
BIORAD
pH meter (Seven Easy)
Mettler Toledo
72
4. Materials and Methods
Phosphorimager screens
Kodak, Fuji
Phosphorimager cassettes
(BAS casette 2040)
Fuji
Phosphorimager (Molecular Imager FX)
BIORAD
Photometer (Carry 100 Bio)
Varian
Photometer (Biophotometer)
Eppendorf
Pipettes (Research)
Eppendorf
Pipette multichannel (Transferpette S-12)
Brand
Pipettes multichannel (SL-Pette)
Süd-Laborbedarf
Pipette (Rainin, edp)
Mettler Toledo
Pipettor
Hirschmann Laborgeräte
Pipetting robot (Microlab STAR)
Hamilton Robotics
Power supply (PowerPack basic)
BIORAD
Power supply (PowerPack 3000)
BIORAD
Reverse osmosis system (arium 61316)
Sartorius
SDS-PAGE system
BIORAD
Sonifier (Sonoplus, UW 2200)
Bandelin
Speed-Vac (Concentrator 5301)
Eppendorf
Sterile hood
HERA safe
Thermocycler
Biometra
Thermomixer (comfort)
Eppendorf
Ultracentrifuge (L-60)
Beckmann Coulter
Ultrapure water system (arium 611)
Sartorius
UV-lamp (HPK-125)
DEMA
Vortexer (Reax 2000, Reax control)
Heidolph
Water bath
Memmert
4.1.17 Disposables
Columns (Empty Reservoir 3 mL, 15 mL)
Biotage
Cuvettes (Plastibrand 1.5 mL)
Brand
384-deepwell plates
Thermo Scientific
96-deepwell plates (2.2 mL)
TreffLab
Electroporation cuvettes (Gene Pulser)
BIORAD
Gloves
MaiMed medical, VWR
Petri dishes
Peske
73
4. Materials and Methods
Sealing foils
4titude,
Sephadex G25 columns
GE Healthcare
Sterile filter paper
Millipore
Syringes
Brand, Henke Sass Wolf
Syringe needles
Braun, Normject
Syringe sterile filters (0.2 µm, 0.45 µm)
Peske
Tips
Axygen
Tips (Qiagen-tip 100)
Qiagen
Tips pipetting robot
Hamilton Robotics
Tips RNase and DNase-free
Sorenson BioScience, Biozym
TLC plates (silica gel 60 F254)
Merck
Tubes (1.5 mL and 2 mL)
Sarstedt, Brand
Tubes (Cap Strips 0.2 mL)
Thermo Scientific
Tubes (PCR 0.2 mL)
TreffLab
Tubes (Falcon 15 mL and 50 mL)
Sarstedt, Peske
Vivaspin ultrafiltration spin columns
Sartorius Stedim Biotech
Whatman chromatography paper (3 mm)
GE Healthcare
96-well filter plates
ABGene
96-well PCR plate (RNaseDNase-free)
TreffLab
96-well plates
Sarstedt
384-well plates (black)
ABGene
4.2
Methods
4.2.1
Chemical synthesis
4.2.1.1 General
All reagents are commercially available and were used without further purification unless
otherwise stated. Triphosphate synthesis was performed under an inert atmosphere. Ion-exchange
chromatography was performed on a DEAE-Sephadex A-25 column and RP-chromatography using a
RP-MPLC (RP-18, 40-63 µm). NMR spectra were recorded on Bruker Avance 400 (1H: 400 MHz,
32P: 162 MHz) spectrometer. The solvent signal was used as reference and the chemical shifts are
reported relative to TMS in ppm (δ). HRMS spectra were recorded on a Bruker mircoTOF II in the
negative mode.
74
4. Materials and Methods
4.2.1.2 Chemical synthesis of 2’-O-methyl-2’-deoxyuridine-5’-triphosphate
The synthesis of the 5’-nucleoside triphosphate of 2’-O-methyl-2’-deoxyuridine was
performed according to a method reported by Kovács and Ötvös.193 40 mg (0.15 mmol) of 2’-Omethyluridine and 66.4 mg (0.31 mmol) proton sponge (1,8-bis(dimethylaminonaphthalene) were
dried for 30 min in vacuo and afterwards dissolved in 1 mL freshly distilled trimethylphosphate. The
reaction mixture was cooled on ice and 25 µL (0.31 mmol) of freshly distilled POCl3 was added. The
reaction was monitored by TLC in isopropanol / water/ ammonia = 3 / 1 / 1. After 15 min, another 25
µL (0.31 mmol) of POCl3 were added to complete the reaction. The reaction was continued stirring for
1 h, then a 0.5 M solution of (Bu3NH)2H2P2O7 in anhydrous DMF (1,5 mL, 0.77 mmol) and 410 µl
(1.5 mmol) tributylamine were added fast and simultaneously. The solution was stirred for 30 min,
afterwards quenched by the addition of 4 ml 0.1 M TEAB buffer pH 7.5 and stirred again for another
30 min. The reaction mixture was extracted three times with 5 mL ethyl acetate, the aqueous phase
evaporated and the residue lyophilized. The compound was further purified by ion exchange
chromatography and the remaining sample desalted by RP-MPLC using 0.05 M TEAA buffer and
acetonitrile to afford the modified 5’-triphosphate (21.18µmol, 14% yield). 1H-NMR (400 MHz, D2O):
δ 1.35 (t, J=7.1 Hz, NCH2CH3); 3.27 (q, J=6.8 Hz, NCH2CH3); 3.58 (s, 3H, OCH3); 4.16 (m, 1H, HC(2')); 4.32 (m, 3H, H-C(4'), H1-C(5'), H2-C(5')); 4.61 (m, 1H, H-C(3')); 6.04 (d, J=8.1 Hz, 1H, HC(5)); 6.11 (d, J=4.2 Hz, 1H, H-C(1')); 8.06 (d, J=8.2 Hz, 1H, H-C(6)) ppm.
31
P-NMR (162 MHz,
D2O): δ -10.00 (d, J=19.7 Hz, 1P, Pγ); -11.50 (d, J=20.1 Hz, 1P, Pα); -23.12 (triplettoid, J=19.3 Hz,
J=20.1 Hz, 1P, Pβ) ppm. HRMS (m/z): [M-H]- calcd for C10H16N2O15P3-: 496.9758; found 496.9754.
4.2.2
Molecular Cloning
4.2.2.1 Polymerase chain reaction (PCR)
Standard PCR reactions using Phusion High Fidelity DNA Polymerase were conducted for the
preparation of double-stranded DNA templates containing the T7 promoter sequence used for in vitro
transcription reactions. For this purpose, forward and reverse primers (0.5 µM each), ssDNA template
(20 nM), dNTPs (200 µM each) and 0.02 U/µL Phusion DNA polymerase were mixed in 1× Phusion
HF buffer in a total reaction volume of 50 µL and amplification performed according to the following
program: 98 °C for 30 s followed by 30 cycles of 98 °C for 10 s, 55 °C for 30 s and 72 °C for 10 s and
a final extension step at 72 °C for 2 min. PCR products were purified by phenol-chloroform-extraction
and ethanol-precipitation.
75
4. Materials and Methods
4.2.2.2 Site-directed mutagenesis
Introduction of single mutations was done according to the QuikChange Site-Directed
Mutagenesis Kit protocol from Stratagene. For this purpose, hole plasmid PCR was performed using
forward and reverse primers containing the desired mutations (0.5 µM each), DNA template (20 ng),
dNTPs (200 µM each), 3-5% DMSO (optional) and 0.02 U/µL Phusion DNA polymerase in 1×
Phusion HF buffer in a total reaction volume of 50 µL. Amplification was performed according to the
following program: 98 °C for 60 s followed by 16-18 cycles of 98 °C for 10 s and 72 °C for 4 min (30
s/kb) and a final extension step at 72 °C for 10 min. PCR product formation was checked by agarose
gel electrophoresis. The methylated parental DNA template was digested by the addition of 20 U Dpn
I and reaction mixtures incubated for 1-2 h at 37 °C. Afterwards, the restriction enzyme was
inactivated at 80 °C for 20 min and 1 µL of the reactions mixture directly used for transformation in E.
coli XL10-Gold cells. Alternatively, reactions mixtures were purified using a MinElute Reaction
Cleanup-Kit (Qiagen) prior to transformation.
4.2.2.3 Multi site-directed mutagenesis
Introduction of 2-4 mutations simultaneously was done using the QuikChange Multi SiteDirected Mutagenesis Kit from Stratagene. Therefore, a single primer containing the desired mutations
is required to mutate each site whereas all primers bind to the same template strand. Reactions
containing each primer (1-3 primers: 100 ng each; 4 primers: 50 ng each), 100 ng DNA template, 1 µl
dNTP-Mix, 1 µL QuikChange Multi enzyme blend and 0.75 µL of QuikSolution in 1× reaction buffer
were mixed in a total reaction volume of 25 µL. Reactions were conducted according to the following
cycling program: 95 °C for 1 min followed by 30 cycles of 95 °C for 1 min, 55 °C for 1 min and 65 °C
for 16 min (2 min/kb). The methylated parental DNA template was digested by the addition of 20 U
Dpn I and reaction mixtures incubated for 1-2 h at 37 °C. Afterwards, the restriction enzyme was
inactivated at 80 °C for 20 min and 1 µL of the reactions mixture directly used for transformation in E.
coli XL10-Gold cells.
4.2.2.4 PCR with randomized primers
For randomizing T7 RNA polymerase at amino acid positions Y639 and H784, two
overlapping DNA fragments (F1: 83 bp and F2: 453 bp) containing each of the randomized regions
were prepared by PCR using oligonucleotide primers in which the codons of amino acid positions
Y639 and H784 were randomized by NNK, whereas N stands for all four bases and K for T and G. A
third unmodified overlapping DNA fragment (F3: 308 bp) was prepared along with these fragments
for subsequent assembly and cloning into pGDR11 using Hind III and Nde I restriction sites. PCR
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4. Materials and Methods
reactions containing primers (FP #1 and RP #2, FP #3 and RP #4, FP #5 and RP #6) (0.5 µM each),
plasmid DNA template (100 pM), dNTPs (200 µM each) and 0.02 U/µL Phusion DNA polymerase in
1× Phusion HF buffer were mixed in a total reaction volume of 50 µL. PCR was accomplished using
the following PCR program: 98 °C for 1 min, 30 cycles of 98 °C for 10 s, 65 °C (F1)/ 60 °C (F2 and
F3) for 15 s and 72 °C for 15 s and a final extension step at 72 °C for 5 min. The PCR products were
purified by preparative agarose gel electrophoresis using 2.5 % TAE agarose.
4.2.2.5 Overlap extension PCR
The overlapping PCR products with randomized positions Y639 and H784 were assembled in
combination with a third unmodified fragment by overlap extension PCR to yield a 806 bp fragment
for cloning using Hind III and Nde I restriction sites. For this purpose, PCR reactions containing the
three overlapping fragments (1 nM each), dNTPs (200 µM), 0.02 U/µL Phusion DNA polymerase in
1× Phusion HF buffer were mixed and PCR accomplished using the following program: 98 °C for 2
min, 10 cycles of 98 °C for 10 s and 72 °C for 1 min. Afterwards, the reaction was cooled to 4 °C and
the two flanking primers (FP#1 and RP#6) were added (0.5 µM each). The reaction was continued
according to the following program: 98 °C for 1 min, 20 cycles of 98 °C for 10 s, 55 °C for 30 s and
72 °C for 25 s (30 s/bp) and a final extension step at 72 °C for 5 min. The assembled 806 bp fragment
was purified by preparative agarose gel electrophoresis using 2.5 % TAE agarose.
4.2.2.6 Error-prone PCR
The construction of a randomized T7 RNA polymerase mutant library starting from mutant
M2 (E593G, Y639V, V685A, H784G) was accomplished by error-prone PCR. The gene of this mutant
was amplified by Taq DNA polymerase under error-prone conditions. For this purpose, plasmid DNA
template (20 pM), primers (0.2 µM each), dCTP/TTP (1 mM), dATP/dGTP (0.2 mM), 0.05 U/µL Taq
DNA polymerase, MgCl2 (7 mM) and MnCl2 (0.1 mM) were mixed in 1× Taq reaction buffer with
KCl in a total reaction volume of 50 µL. Amplification was performed in a 2-step protocol according
to the following program: 95 °C for 2 min, 15 cycles of 95 °C for 1 min and 72 °C for 4 min and a
final extension step at 72 °C for 5 min. Afterwards, the randomized PCR products were purified by
preparative agarose gel electrophoresis using 0.8 % TAE agarose.
4.2.2.7 Restriction digest of double-stranded DNA
PCR products and plasmid DNA were digested with the respective restriction endonucleases
according to the manufacturer’s instructions. For this purpose, about 1 µg of DNA was incubated with
1 µL of each restriction enzyme for 3 h at 37 °C in the respective 1× reaction buffer in a final reaction
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4. Materials and Methods
volume of 50 µl. The digest was followed by a heat inactivation step at 65 °C or 80 °C for 20 min. The
resulting DNA was further purified by preparative agarose gel electrophoresis or by use of a MinElute
Reaction Cleanup Kit. Plasmid DNA was dephosphorylated prior to purification.
4.2.2.8 Dephosphorylation of double-stranded DNA
5’-Dephosphorylation of digested plasmid DNA was accomplished by the addition of 1 µl
Antarctic Phosphatase (5 U/µL), 3 µL ddH2O and 6 µL 10× Antarctic reaction buffer directly to the
restriction digest (50µL). The reaction mixture was incubated for 1 h at 37 °C and the phosphatase
afterwards inactivated by incubation at 65 °C for 5 min. The dephosphorylated plasmid DNA was
purified by preparative agarose gel electrophoresis using 0.8 % TAE agarose.
4.2.2.9 DNA Ligation
Ligation of pre-cut DNA insert into pre-cut and dephosphorylated plasmid DNA was
accomplished by incubating 30-50 ng of the plasmid with a 3 or 5 fold molar excess of insert in the
presence of 1 µL T4 DNA ligase in 1× reaction buffer at 16 °C for 16 h in a total reaction volume of
20 µL. Afterwards, the ligase was inactivated by incubation at 65 °C for 15 min and ligation reactions
stored at -20 °C. Ligation control reactions were conducted without the addition of DNA insert.
Ligation reactions were directly used for E. coli transformation or prior to that purified by use of a
reaction cleanup kit.
4.2.2.10 Sequencing of double-stranded DNA
Sequencing of plasmid DNA isolated from E. coli liquid cultures was done at GATC Biotech.
For this purpose, the DNA was diluted to a final concentration of 30-100 ng/µl in a final volume of 30
µL.
4.2.2.11 Analytical agarose gel electrophoresis
To visualize DNA fragments of different sizes, DNA samples were mixed with an appropriate
volume of 6× DNA loading dye and separated by electrophoresis using 0.8% or 2.5% agarose gels in
0.5× TBE buffer by applying 120 V. The agarose gel was stained in 0.5× TBE buffer supplemented
with ethidium bromide for 15 min. After destaining the gel for another 15 min in 0.5× TBE buffer,
DNA fragments were visualized under UV light (302 nm).
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4. Materials and Methods
4.2.2.12 Preparative agarose gel electrophoresis
To purify DNA fragments of different sizes, DNA samples were mixed with an appropriate
volume of 6× DNA loading dye and separated by electrophoresis using 0.8% or 2.5% agarose gels in
1× TAE buffer by applying 100 V. The agarose gel was stained in 1× TAE buffer supplemented with
ethidium bromide for 15 min. After destaining the gel for another 15 min in 1× TAE buffer, DNA
fragments were visualized under preparative UV light (302 nm) and bands excised with a scalpel. The
DNA was isolated from the agarose gel using a gel extraction kit following the manufacturers
instructions.
4.2.2.13 SDS-PAGE
To separate and visualize proteins with different sizes, discontinuous SDS-PAGE according to
Leammli with a 4% stacking gel and a 12% separating gel was used. Proteins were mixed with an
appropriate volume of 6× SDS loading dye and incubated at 95 °C for 5 min. Afterwards, the protein
samples were loaded on the gel and proteins separated by electrophoresis in 1× SDS-PAGE running
buffer at 30-45 mA. The SDS-PAGE gel was stained using coomassie brilliant blue staining solution.
4.2.2.14 Denaturing PAGE
Denaturing PAGE was used to separate DNA and RNA oligonucleotides according to their
sizes. RNA samples resulting from in vitro transcription were mixed with an equal volume of
denaturing PAGE loading dye, DNA samples resulting from primer extension reactions were mixed
with a 2.25 fold volume of denaturing PAGE loading dye. DNA samples were incubated at 95 °C for 5
min prior to loading. Depending on the oligonucleotide fragments, 8-12% denaturing PAGE gels were
used and oligonucleotides separated in 1× TBE buffer by applying 100 W and 3000 V at 45 °C. The
resulting gel was transferred to a whatman paper, dried in vacuo at 80 °C for 30-60 min and afterwards
exposed to a phosphor imager screen overnight. Visualization of radioactively labeled oligonucletides
was done by phosphorimaging.
4.2.2.15 Preparative denaturing PAGE
Purification of single stranded DNA and RNA oligonucleotides was done by preparative
denaturing PAGE using 1.5 mm thick 8-12 % denaturing PAGE gels. DNA or RNA samples were
mixed with denaturing PAGE loading dye, loaded on the gel and fragments separated in 1× TBE
buffer by applying 100 W and 800 V at 45 °C for about 3 h. Afterwards, DNA or RNA
oligonucleotides were visualized under UV light (254 nm) using TLC plates coated with a
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4. Materials and Methods
fluorescence indicator and bands excised using a scalpel. The excised bands were crushed and DNA
eluted into ddH2O at 45°C and RNA at 4°C overnight. Finally, oligonucleotides were ethanol
precipitated, dissolved in ddH2O and concentrations determined by absorption measurements at 260
nm.
4.2.3
Oligonucleotide based methods
4.2.3.1 Dephosphorylation of ssRNA ladder
Low Range ssRNA ladder is a RNA standard composed of 6 RNA molecules of different sizes
deriving from in vitro transcription reactions. For efficient 5’-radioactive labeling of the RNA ladder
using [γ-32P]ATP in forward reaction with T4 Polynucleotide Kinase (PNK), 5'-phosphate groups from
RNA were removed using calf intestinal alkaline phosphatase (CIAP). For this purpose, 200 ng/µL
ssRNA ladder, 0.1 U/µL CIAP and 1 U/µL RiboLock were incubated in 1× CIAP reaction buffer for
30 min at 37°C in a total reaction volume of 20 µL. The reaction was stopped by phenol-chloroform
extraction and the RNA purified by ethanol precipitation.
4.2.3.2 5’-Phosphorylation of oligonucleotides using [γ-32P]ATP
DNA
DNA oligonucleotide primers were radioactively labeled using [γ-32P]ATP and T4
polynucleotide kinase (PNK). For this purpose, the DNA oligonucleotide (400 nM), 400 nCi/µL [γ32
P]ATP and 0.4 U/µL T4 PNK were incubated in 1× PNK reaction buffer A for 1 h at 37°C in a total
reaction volume of 50 µL. The reaction was stopped by incubating the mixture for 2 min at 95°C. The
radioactively labeled oligonucleotide was purified using a Sephadex G25 spin column. For primer
extension reactions, the labeled primer was diluted with unlabeled primer to obtain a final
concentration of 3 µM (70 µL).
RNA
The ssRNA ladder was radioactively labeled using [γ-32P]ATP and T4 polynucleotide kinase
(PNK). For this purpose, 50 ng/µL dephosphorylated RNA standard was incubated in the presence of
500 nCi/µL [γ-32P]ATP, 0.5 U/µL T4 PNK, 1 U/µL RiboLock in 1× reaction buffer A for 30 min at
37°C in a total reaction volume of 20 µL. The reaction was stopped by phenol-chloroform extraction
and the RNA ladder purified using a Sephadex G25 spin column.
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4. Materials and Methods
4.2.3.3 Ethanol precipitation
For DNA and RNA precipitation, the sample was mixed with 1/10 volume of 3 M NaOAc pH
5.2 and 2.5 volumes of ice-cold 100% ethanol. After incubating the mixture for ≥ 2 h at -20 °C, the
sample was centrifuged for 30 min at 4 °C (20.000 ×g). The supernatant was discarded and the
resulting pellet washed with 70% ice-cold ethanol. The sample was again centrifuged for 15 min at 4
°C (20.000 ×g) and the washing step repeated. The resulting DNA/RNA pellet was dried in vacuo,
resolved in ddH2O and stored at -20 °C.
4.2.3.4 Phenol-chloroform extraction
To remove proteins from DNA or RNA containing samples, the solution was mixed with an
equal volume of phenol-chloroform-isoamyl alcohol and centrifuged for 2 min at room temperature
(13.400 rpm). The upper aqueous phase was saved and twice extracted with an equal volume of
chloroform-isoamyl alcohol. Afterwards, the DNA/RNA was precipitated using ethanol precipitation.
4.2.3.5 Electrospray ionization MS (ESI-MS) of oligonucleotides
To check masses of DNA oligonucleotides by ESI-MS, the DNA sample was diluted in 20%
isopropanol/1% NEt3 to a final concentration of 5 µM and directly injected.
4.2.3.6 Determination of DNA and RNA concentration
DNA and RNA concentrations were determined by absorption measurements at 260 nm using
a spectrophotometer and the corresponding calculated molar extinction coefficient. The concentration
of plasmid DNA was exclusively determined with a Nanodrop spectrophotometer.
4.2.4
Microbiological methods
4.2.4.1 Preparation of electrocompetent E. coli cells
For the preparation of electrocompetent E. coli cells 500 mL of LB-medium was inoculated
with 2 mL of an overnight culture of the desired E. coli strain and grown at 25 °C and 200 rpm to an
OD600 of 0.6-0.7. Cells were incubated on ice for 15 min and afterwards harvested by centrifugation
(4.500 x g for 20 min, 4 °C). All further steps were conducted on ice under a sterile hood. The
supernatant was discarded and the cell pellet resuspended in 500 mL of sterile precooled ddH2O. After
centrifugation (4.500 x g for 20 min, 4 °C), this washing step was repeated once. Afterwards, the pellet
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4. Materials and Methods
was resuspended in 50 mL sterile precooled 10 % glycerol and again centrifuged (4.500 x g for 10
min, 4 °C). The supernatant was removed and the cell pellet resuspended in 1 mL sterile precooled 10
% glycerol. Aliquots of 80 µL were directly used for transformation and the remaining aliquots frozen
in liquid nitrogen and stored at -80 °C until further use.
4.2.4.2 Transformation of electrocompetent E. coli cells
Transformation of electrocompetent E. coli cells was performed by mixing 1-2 µL of ligation
mixture or 1 ng of plasmid DNA with 80 µL of electrocompetent E. coli cells on ice in a Gene Pulser
electroporation cuvette (1 mm). Transformation was accomplished by applying 1800 V and cells were
subsequently mixed with 1 mL preheated SOC medium. The cell suspension was incubated at 37 °C
for 45 min shaking. Afterwards, 50-150 µL of the cells or the entire transformation mixture were used
for LB agar plate cultures.
4.2.4.3 LB agar plate cultures
E. coli cells containing plasmid DNA were grown on LB agar plates supplemented with the
respective antibiotic for selection. For this purpose, 50-150 µL of the transformation mixture was
plated on LB agar plates under a sterile hood and incubated at 37 °C overnight. Cells from a glycerol
stock were also cultivated on LB agar plates prior to liquid culture preparation if required by plating a
small amount of cells from the frozen stock.
4.2.4.4 Liquid cultures
Overnight liquid cultures were prepared by inoculating 5-50 mL LB medium supplemented
with the respective antibiotic for selection with E. coli cells from a single colony grown freshly on a
LB agar plate. The liquid cultures were incubated overnight at 37 °C and 200 rpm shaking.
Alternatively, liquid cultures were inoculated with cells from an E. coli glycerol stock.
4.2.4.5 Plasmid DNA isolation from liquid cultures
Isolation of plasmid DNA from 2-4 ml E. coli overnight liquid culture was done according to
the manufacturer’s instructions using a plasmid Miniprep kit. The isolation of high amounts of
ultrapure plasmid DNA from 25 mL of E. coli overnight liquid culture was done according to the
manufacturer’s instructions using a Midiprep Kit and QIAGEN-tip 100 columns.
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4. Materials and Methods
4.2.4.6 Colony PCR
Colony PCR was used to analyze cloning products in E. coli single colonies. For this purpose,
several single colonies were picked from a freshly grown LB agar plate, dissolved in 10 µL of ddH2O
each and these suspensions used as templates in standard PCR reactions using Taq DNA polymerase.
The PCR mixture was composed of 1 µL bacterial suspension, primers (0.2 µM), dNTPs (200 µM
each), 0.05 U/µL Taq DNA polymerase and 1 × KTQ reaction buffer in a final reaction volume of 50
µL. Amplification was conducted using the following program: 95 °C for 5 min, followed by 30
cycles of 95 °C for 1 min, 65 °C for 30 s and 72 °C for 3 min and a final extension step at 72 °C for 5
min. PCR products were analyzed by agarose gel electrophoresis.
4.2.4.7 E. coli glycerol stock preparation
E. coli cells containg plasmid DNA were mixed in a 1:1 ratio with 50 % glycerol in LB
medium supplemented with the respective antibiotic and afterwards shock frozen in liquid nitrogen for
long term storage at -80 °C. Glycerol stocks of E. coli BL21 cells containing the T7 RNA polymerase
mutant libraries were prepared in 384 deepwell plates. For this purpose, single colonies were picked
and cultivated in 384 deepwell plated containing 150 µL/ well LB medium supplemented with the
respective antibiotic at 25 °C shaking overnight. 100 µL/ well glycerol was added to a final
concentration of 20 % (v/v) for long term storage at –80 °C.
4.2.5
Biochemical methods
4.2.5.1 Expression of T7 RNA polymerases
E. coli BL21 (DE3) cells were transformed with pGDR11 containing the respective T7 RNA
polymerase gene. Expression of T7 RNA polymerase wildtype, mutant M1 (Y639F, H784) and mutant
M2 (E593G, Y639V, V685A, H784G) was performed by inoculating 1 l LB-medium supplemented
with 100 µg/ml carbenicillin with an E. coli overnight liquid culture (1:100). Cells were grown at 37
°C and 200 rpm to an OD600 of 0.6-0.7 and expression was induced by adding IPTG to a final
concentration of 1 mM. After 4 h of expression, cells were harvested by centrifugation (4.500 × g for
20 min, 4 °C) and pellets stored at -80°C until further use.
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4. Materials and Methods
4.2.5.2 Purification of T7 RNA polymerases
Purification of the polymerases bearing an N-terminal polyhistidine (6 × His) tag was
performed by nickel affinity chromatography using Ni-IDA chelating sepharose on an ÄKTA Purifier
FPLC system. Therefore, the cell pellets were resuspended in 50 ml T7 RNAP lysis buffer and
incubated on ice for 30 min. Cells were disrupted by sonification and lysates cleared by centrifugation
(22.000 × g for 30 min, 4 °C). The supernatant was loaded on a Ni-IDA column equilibrated with 3 %
T7 RNAP IMAC buffer B and proteins eluted by gradient-elution with 100 % T7 RNAP IMAC buffer
B. Fractions containing the purified protein were pooled and the buffer exchanged with 2 × storage
buffer using a Vivaspin 50.000 MWCO concentrator. Glycerol was added to a final concentration of
50% (v/v) and proteins stored at -20 °C.
4.2.5.3 Expression of T7 RNA polymerase mutant libraries
E. coli BL21 cells were transformed with pGDR11 containing the mutated T7 RNA
polymerase genes. T7 RNA polymerase variants from the mutant libraries were expressed in parallel
in 96-deepwell plates for 5 h at 37 °C. For this purpose, 1 ml LB-medium supplemented with 100
µg/ml carbenicillin was inoculated with 10 µL of the 384-deepwell library stock using a pipetting
robot. Afterwards, cells were grown to an OD600 of 0.6-0.7 and expression induced by the addition of
IPTG to a final concentration of 1 mM. Cells were harvested by centrifugation (4.500 × g for 20 min,
4 °C) and pellets stored at -80 °C for further processing.
4.2.5.4 Preparation of E. coli BL21 lysates for screening of T7 RNA polymerase mutant
libraries
After protein expression in 96-deepwell plates, cells were resuspended and lysed by the
addition of 300 µl/well screening lysis buffer and incubated for 20 min at 25°C. Afterwards, lysates
were diluted by the addition of precooled 1.2 mL/well screening dilution buffer, mixed and cleared by
centrifugation (4.500 × g for 45 min, 4 °C). Supernatants containing T7 RNA polymerase mutants
were directly used for screening.
4.2.5.5 Purification of T7 RNA polymerase hits in 96-well filter plates
The 55 identified hits from the T7 RNA polymerase mutant library in which amino acid
positions Y639 and H784 were randomized were purified and adjusted to the same concentration prior
to characterization. For this purpose, hits were initially expressed in 1 mL LB-medium in 96-deepwell
plates as described above. Afterwards, cells were resuspended and lysed by the addition of 500 µl/well
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4. Materials and Methods
T7 RNAP lysis buffer and incubated for 20 min at 25 °C. The lysates were centrifuged (4.500 × g for
30 min, 4 °C) and the supernatants transferred to a 96-well filter plate filled with 25 µl/well Ni-IDA
sepharose. The 96-well plate was centrifuged (500 rpm for 30 s, 4 °C), the flow through discarded and
the Ni-IDA matrix washed twice with 400 µl/well T7 RNAP IMAC binding buffer and twice with 400
µl/well T7 RNAP IMAC washing buffer. Proteins were eluted by the addition of 50 µl/well T7 RNAP
IMAC buffer B and DTT and EDTA added to a final concentration of 5 mM and 1 mM, respectively,
by the addition of 1/10 volume of 20 × storage buffer. For storage at -20 °C, glycerol was added to a
final concentration of 50 %.
4.2.5.6 Expression and purification of T7 RNA polymerase hits
Hits from the T7 RNA polymerase mutant libraries were purified and adjusted to the same
concentration prior to characterization. For this purpose, hits were expressed in medium scale by
inoculating 50-500 mL LB-medium supplemented with 100 µg/ml carbenicillin with an E. coli BL21
overnight liquid culture (1:100). Cells were grown at 37 °C and 200 rpm to an OD600 of 0.6-0.7 and
expression was induced by adding IPTG to a final concentration of 1 mM. After 5 h of expression,
cells were harvested by centrifugation (4.500 × g for 20 min, 4 °C) and pellets stored at -80°C. For
purification, cells were resuspended and lysed by the addition of 1/20 volume of T7 RNAP lysis buffer
and incubated on ice for 30 min. Afterwards, cells were disrupted by sonification and lysates
centrifuged (22.000 × g for 30 min, 4 °C). Supernatants were loaded on Ni-IDA sepharose gravity
flow columns and the flow through discarded. The matrix was twice washed with 20 column volumes
of T7 RNAP IMAC binding buffer and twice with 20 column volumes of T7 RNAP IMAC washing
buffer. Proteins were eluted by the addition of 10 column volumes of T7 RNAP IMAC buffer B and
the buffer exchanged with 2 × T7 RNAP storage buffer using a Vivaspin 30.000 MWCO concentrator.
Glycerol was added to a final concentration of 50% (v/v) and proteins stored at -20 °C.
4.2.5.7 Expression of KOD DNA polymerases
The E. coli codon optimized gene of KOD (exo-) DNA polymerase in pET24a was ordered
from Geneart (Regensburg, Germany). The mutants KOD (exo-) L408Q and KOD (exo-) L408Q,
A485L were generated by multi site-directed mutagenesis and site-directed mutagenesis according to
(4.2.2.2) and (4.2.2.3). E. coli BL21 (DE3) cells were transformed with the respective plasmid
containing a polyhistidine (6 × His) tag at the C-terminus of the polymerase gene for protein
purification. For each DNA polymerase, expression was performed by inoculating 1 l LB-medium
supplemented with 34 µg/ml kanamycin with an E. coli overnight liquid culture (1:100). Cells were
grown at 37 °C and 200 rpm to an OD600 of 0.6-0.7 and expression was induced by addition of IPTG
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4. Materials and Methods
to a final concentration of 1 mM. After 2.5 h of expression, cells were harvested by centrifugation
(4.500 × g for 20 min, 4 °C) and pellets stored at -80°C until further use.
4.2.5.8 Purification of KOD DNA polymerases
Purification of KOD (exo-) DNA polymerase and mutants was performed by immobilized
metal ion affinity chromatography using Ni-IDA chelating sepharose on an ÄKTA Purifier FPLC
system. For this purpose, the cell pellet was resuspended in 50 ml KOD lysis buffer and incubated at
37 °C for 20 min. Cells were disrupted by sonification, E. coli host proteins denatured at 85 °C for 10
min and lysates cleared by centrifugation (146.000 × g for 1 h, 4 °C). The supernatant was loaded on a
Ni-IDA column equilibrated with 2 % KOD IMAC buffer B and proteins eluted by gradient-elution
with 100 % KOD IMAC buffer B. Fractions containing the purified protein were pooled and the buffer
exchanged with 2 × storage buffer using a Vivaspin 50.000 MWCO concentrator. For storage, 50%
(v/v) glycerol and Triton X-100 to a final concentration of 0.1% (v/v) were added.
4.2.5.9 Determination of protein concentrations via Bradford protein assay
Concentrations of proteins that were identified by SDS-PAGE to be ≥95 % pure were
determined via Bradford protein assay. For this purpose, 20 µl of different protein dilutions and a
BSA-standard (0.5 mg/ml – 15.625 µg/ml) were prepared in the corresponding 1 × storage buffer and
each mixed with 1 mL of a 1 × RotiQuant solution in a disposable cuvette. After an incubation time of
10 min at room temperature, the absorption at 595 nm was measured with a UV-spectrophotometer.
Protein concentrations were determined by use of the BSA standard curve.
4.2.6
Assays
4.2.6.1 In vitro-transcription assays
Transcription reactions using NTPs and 2’-modified NTPs were performed for 1 - 2 h at 37 °C
in a total reaction volume of 20 µL. The reaction mixtures contained 0.1 - 0.5 µM DNA template, 1
mM NTPs and 200-500 nM T7 RNA polymerase in 1 × transcription buffer. DNA templates were
annealed with a short non-template DNA strand to form a double-stranded T7 promoter region by
incubating the oligonucleotides at 95 °C for 3 min and subsequently cooling to 25°C (0.5 °C/s) in 1 ×
transcription. If double-stranded DNA templates containing the T7 promoter sequence were used,
those were prepared by PCR using Phusion DNA polymerase and purified by phenol-chloroform
extraction and ethanol precipitation. Reactions were initiated by the addition of the T7 RNA
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4. Materials and Methods
polymerase and transcripts body labeled by inclusion of 0.75 - 1 % (v/v) 3000 Ci/mmol [α-32P]GTP.
Reactions were stopped by adding an equal volume of denaturing PAGE loading dye and transcripts
analyzed by electrophoresis on 10-12% denaturing PAGE gels and phosphorimaging.
4.2.6.2 Time course experiment for the incorporation of 2’-SeCH3-NTPs
Transcription reactions were performed as described above. Aliquots (2 µL) were withdrawn
from the reaction mixture at the corresponding time points and added to an equal volume of denaturing
PAGE loading dye and cooled on ice. Transcripts were analyzed by electrophoresis on 10%
denaturing polyacrylamide gels and visualized by phosphorimaging.
4.2.6.3 Quantification of 2’-SeCH3-modified RNA
RNA quantification was accomplished by in vitro transcription using 0.5 µM DNA template, 1
mM NTPs and 500 nM T7 RNA polymerase in 1 × transcription buffer in a total reaction volume of
400 µL. Transcripts were body labeled by the addition of 1 % (v/v) 3000 Ci/mmol [α-32P]GTP. After
an incubation time of 2 h at 37 °C, reactions were stopped by the addition of 0.125 U/µl DNase I and
DNA templates digested for 15 min at 37 °C. RNA transcripts were precipitated with EtOH at –20 °C
over night. The precipitate was dissolved in equal volumes of ddH2O and denaturing PAGE loading
dye and RNA purified by electrophoresis on 8% preparative denaturing PAGE gels. The excised bands
were crushed and the RNA eluted into ddH2O at 4 °C over night. After EtOH precipitation, the RNA
was dissolved in ddH2O and concentrations measured using a BioPhotometer at 260 nm.
4.2.6.4 T7 RNA polymerase screening assay
Transcription reactions for screening were performed in 384-well plates in reaction volumes of
20 µl. The reaction mixture contained 0.2 µM DNA template, 1 mM NTPs, 5 µL of RNA polymerase
containing lysate and 1× transcription buffer. After an incubation time of 90 min at 37 °C, DNA
templates were digested by the addition of 5 µL DNase I (0.4 U/µl). Digestion was performed for 30
min at 37 °C and the reactions stopped by the addition of 45 µl/well SYBR Green II stop solution.
After an incubation time of 5 min at room temperature, fluorescence intensities were determined by
fluorescence readout using a microplate reader with an excitation wavelength of 485 nm and an
emission wavelength of 520 nm.
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4. Materials and Methods
4.2.6.5 Primer extension reactions using araTTP, hTTP, xyloTTP and ceTTP
Primer extension reactions contained 150 nM
32
P-labeled primer (BRAF-23C), 225 nM of
template (69-PolyT, 69-PolyA), 200 µM of modified or unmodified TTP, 50-250 nM of DNA
polymerase and 1 × reaction buffer in a total reaction volume of 20 µl. Primer and template were
annealed in the absence of dNTPs and DNA polymerase by incubating the mixture at 95 °C for 3 min
and subsequent cooling to 25 °C (0.5 °C/s). Reactions were started by the addition of dNTPs or DNA
polymerase and incubated for 30-180 min at adequate temperatures (human DNA polymerase β and
Dpo4 DNA polymerase: 37 °C; Therminator DNA polymerase: 55 °C; KlenTaq, Taq and 9°North
exo(-) DNA polymerase: 72 °C). Reactions were stopped by the addition of 40 µL denaturing PAGE
loading dye (stop solution), denatured for 5 min at 95 °C and analyzed by 12% denaturing PAGE.
4.2.6.6 Primer extension reactions using xyloTTP and xylo-dATP
Primer extension reactions contained 150 nM
32
P-labeled primer (BRAF-23C), 225 nM of
template (69-PolyT, 69-PolyA), 200 µM of modified or unmodified TTP/dATP, 100 nM of DNA
polymerase and 1 × reaction buffer in a total reaction volume of 20 µl. Primer and template were
annealed in the absence of dNTPs and DNA polymerase by incubating the mixture at 95 °C for 3 min
and subsequent cooling to 25 °C (0.5 °C/s). Reactions were started by the addition of dNTPs and
incubated for 60 min at adequate temperatures (human DNA polymerase beta and Dpo4 DNA
polymerase: 37 °C; Therminator and 9°North exo(-) DNA polymerase: 55 °C; KlenTaq and Taq DNA
polymerase: 72 °C). Reactions were stopped by the addition of 40 µL denaturing PAGE loading dye
(stop solution), denatured for 5 min at 95 °C and analyzed by 12% denaturing PAGE.
88
5. Appendix
5 Appendix
5.1
Sequences
5.1.1
Oligonucleotide primer and templates
All used oligonucleotide primers and templates were 2’-deoxynucleotides.
5.1.1.1 Primer used for site-directed-mutagenesis
VS_Sousa_Y639F_FP
5’-GCGTTATGACCCTGGCCTTTGGTAGCAAAGAATTTGG-3’
VS_Sousa_Y639F_RP
5’-CCAAATTCTTTGCTACCAAAGGCCAGGGTCATAACGC-3’
VS_Sousa_H784A _FP
5’-GGTATTGCACCGAATTTTGTGGCTAGCCAGGATGGTAG
CCATC-3’
VS_Sousa_H784A _RP
5’-GATGGCTACCATCCTGGCTAGCCACAAAATTCGGTGCAA
TACC-3’
VS_RGVG_ Y639V
5’-GTAGCGTTATGACCCTGGCCGTTGGTAGCAAAGAA
TTTGG-3’
VS_RGVG_ H784G
5’-GTATTGCACCGAATTTTGTGGGTAGCCAGGATGGTA
GCC-3’
VS_RGVG_ E593G
5’-CAATTAATGGCACCGATAATGGCGTTGTTACCGTGAC
CGATG-3’
VS_RGVG_ V685A
5’-CTGATTTGGGAAAGCGCGAGCGTTACCGTTGTTGCAG-3’
SDM_NdeI_pGDR11_FP
5’-GATTGTACTGAGAGTGCACCTCATGCGGTGTGAAATA
CCGC-3’
SDM_NdeI_pGDR11_RP
5’-GCGGTATTTCACACCGCATGAGGTGCACTCTCAGTAC
AATC-3’
KODexo- pET24a L408Q
5’-CTGGATTTTCGCAGCCAGTATCCGAGCATTATTATTA
CC-3’
KODexo- pET24a *775L
5’-GAAACCGAAAGGCACCTTACTCGAGGGCAGCG-3’
KODexo- pET24a *791S
5’-CTGTATTTTCAGGGCTCAAAGCTTGCGGCCGC-3’
KODexo(-) pET24a A485L FP
5’-CTGCTGGATTATCGTCAGCGTCTGATTAAAATTCTGG
CCAATAG-3’
KODexo(-) pET24a A485L RP
5’-CTATTGGCCAGAATTTTAATCAGACGCTGACGATAA
TCCAGCAG-3’
KOD A485L SDM1 FP
5’-CTGCTGGATTATCGTCAGCGTCTAATTAAAATTCTGG
CCAATAG-3’
89
5. Appendix
KOD A485L SDM1 RP
5’-CTATTGGCCAGAATTTTAATTAGACGCTGACGATAA
TCCAGCAG-3’
5.1.1.2 Primer used for cloning and PCR
M = 50.8% A / 49.2% C N = 30.1% A / 19.6% G / 29.2% C / 21.1% T
T7 RNAP NNK-Bib FP #1
5’-GGCTGGCATATGGTGTTACCC-3’
T7 RNAP NNK-Bib RP #2
5’-GACGAAAACCAAATTCTTTGCTACCMNN
GGCCAGGGTCATAACGCTAC-3’
T7 RNAP NNK-Bib FP #3
5’-GCAAAGAATTTGGTTTTCGTC-3’
T7 RNAP NNK-Bib RP #4
5’-CGCAGATGGCTACCATCCTGGCTMNN
CACAAAATTCGGTGCAATAC-3’
T7 RNAP NNK-Bib FP #5
5’-CAGGATGGTAGCCATCTGCG-3’
T7 RNAP NNK-Bib RP #6
5’-CTAATTAAGCTTTTAGGCAAAGG-3’
T7 RNAP EP-PCR RP
5’-CAGGAGTCCAAGCTCAGCTAATTAAGCTTTTA-3’
T7 RNAP EP-PCR FP
5’-GGATCCGCATGCAATTGAAGGTCGCATGAATACC-3’
5.1.1.3 Oligonucleotides used for in vitro transcription
Templates for in vitro transcription reactions containing a double-stranded T7 promotor sequence were
either prepared by annealing the template strand with the T7 Promotor sequence or by PCR.
T7 Promotor + 6nt + GGG
5’-GCCTCCTAATACGACTCACTATAGGG-3’
T7 Promotor + 6nt
5’-GCCTCCTAATACGACTCACTATA-3’
T7Pro HIVTAR Hairpin
5’-ACGCACGCTGTAATACGACTCACTATA-3’
Templat HIVTAR Hairpin
5’-GGCAGAGAGCCGAAGCTCAGATCTGCCTATAGTGAGTCG
TATTACAGCGTGCGT-3’
Templat sarcin-ricin loop + T7P
5’-CACTCCGGTCCTCTCGTACTAGGAGCATATAGTGAGTCG
TATTAGGAGGC-3’
E.coli S23rRNA33-mer
2644-2676
5’-GTCCACTCCGGTCCTCTCGTACTAGGAGCAGCCTATAGTG
AGTCGTATTAGGAGGC-3’
E.coli S23rRNA35-mer
2643-2677
5’-CGTCCACTCCGGTCCTCTCGTACTAGGAGCAGCCCTATAG
TGAGTCGTATTAGGAGGC-3’
90
5. Appendix
Templat human SRP RNA
5’-GGTGACCTCCCGGGAGCGGGGGACCACTAGCTCTAGAGA
TAGAGCCTGATGAGTCCGTGAGGACGAAAGTGGTAAGCT-3’
P1-human SRP RNA T7P
5’-GCCTCCTAATACGACTCACTATAGGTGACCTCCCGGGAG
CGGG-3’
P2-human SRP RNA RP
5’-AGCTTACCACTTTCGTCCTC-3’
DNA template 112 nt mod. UTP
5’-CGTTGGTCCTGAAGGAGGATAGGTTGATTTTCTGCAGTCC
TCTGTCCACGGCGGCGCGTGCTGCGCGACGGCACAGCTGACGGTC
TCCCTATAGTGAGTCGTATTAGGAGGC-3’
F20
5’-CGTTGGTCCTGAAGGAGGAT-3’
DNA template 112 nt mod. CTP
5’-CATTAATCCTAGGAAGAAGTGAATTAGTTTTCTACGATCC
TCTATCCGCAACAACACATACTACACAGCAACGCGAC
TAGCAATCTCCCTATAGTGAGTCGTATTAGGAGGC-3’
FP 112 nt mod. CTP
5’-CATTAATCCTAGGAAGAAGT-3’
5.1.1.4 Primer and templates used for primer extension reactions
Primer BRAF 23C
5’-GAC CCA CTC CAT CGA GAT TTC TC-3’
69-PolyA (46A)
3’-CTGGGTGAGGTAGCTCTAAAGAGAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAA-5’
69-PolyT (46T)
3’-CTGGGTGAGGTAGCTCTAAAGAGTTTTTTTTTTTTTTTTTTTTTT
TTTTTTTTTTTTTTTTTTTTTTTT-5‘
5.1.1.5 Primer used for DNA sequencing
pQE-FP-30
5’-TTATTTGCTTTGTGAGCGG-3’
pQE-RP-50
5’-CTAGCTTGGATTCTCACC-3’
SeqPrimer1-T7 RNAP in pGDR11
5’-GGCAAATGGTCGTCGTCCGC-3’
T7
5’- TAATACGACTCACTATAGGG-3’
pET24a
5’-GGGTTATGCTAGTTATTGCTCAG-3’
KOD-exominus-pET24a Seq Primer
5’-GCACTGGGTCGTGATGGTAG-3’
91
5. Appendix
5.1.2
Plasmids
5.1.2.1 pGDR11
The pGDR11 vector is a derivative of the pQE31 vector with additional laqIq gene.189
CTCGAGAAATCATAAAAAATTTATTTGCTTTGTGAGCGGATAACAATTATAATAGATTCAATTGTGAGCGGATAACAATT
TCACACAGAATTCATTAAAGAGGAGAAATTAACTATGAGAGGATCTCACCATCACCATCACCATACGGATCCGCATGCGA
GCTCGGTACCCCGGGTCGACCTGCAGCCAAGCTTAATTAGCTGAGCTTGGACTCCTGTTGATAGATCCAGTAATGACCTC
AGAACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATTGGTGAGAATCCAAGCTAGCTTGGCG
AGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCG
TAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTT
TAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCG
GAATTTCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCA
AACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGG
CGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTG
AGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATAC
GCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGC
TTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATTTTTTTAAGGCAGTTATTGGTGCCCTTAAACG
CCTGGGGTAATGACTCTCTAGCTTGAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCT
GTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGCTCTAGATTACCGTGCAGTCGATGATAAGCTGTCAA
ACATGAGAATTGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACC
TGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCT
TTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGG
TTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTAT
CCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTT
GGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGT
CGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAG
ACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACC
GTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGG
CAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTG
TGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCG
AGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTT
TGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTC
GCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAA
CGTTACTGGTTTCATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCACCAT
TCGATGGTGTCGGAATTTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCTAGAGCTGCCTCGCGCGTTTCGGT
GATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACA
AGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGT
ATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCG
TAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCTGTCGGCTGCGGCGA
GCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAA
AGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATC
ACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCC
CTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTC
TCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTC
AGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCA
GCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTA
CACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCG
GCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAA
GATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATC
AAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGT
CTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGCTGCCTGACTC
CCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTC
ACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCT
CCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATT
GCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTAC
ATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGT
TATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAG
TACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGC
GCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGT
TGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGA
GCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTT
TCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAA
TAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAA
AATAGGCGTATCACGAGGCCCTTTCGTCTTCAC
92
5. Appendix
5.1.2.2 pET24a (+)
ATCCGGATATAGTTCCTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTATTGC
TCAGCGGTGGCAGCAGCCAACTCAGCTTCCTTTCGGGCTTTGTTAGCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTC
GAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGGATCCGCGACCCATTTGCTGTCCACCAGTCATGCTAGCCATA
TGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTGTTATCCGCTCACAATTCCCCTATAGTGAGT
CGTATTAATTTCGCGGGATCGAGATCTCGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCG
GTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGG
CGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGG
TGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAGATCCCGGACAC
CATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAAC
CAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCAC
GTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACT
GGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGA
TTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCG
GCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGT
GGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCC
ATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTA
AGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACG
GGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGC
TGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCG
GTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCT
GGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCAC
TGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTG
GCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTAAGTTAGCTCACTCATTAGGCACCGG
GATCTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCA
CTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTT
TCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTG
GTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGCATGGCGGCCCCACGGGTGCGCATGATCGTGCTC
CTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTG
AAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGG
AAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCT
ACATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCCATACCGCCAGTTGTTT
ACCCTCACAACGTTCCAGTAACCGGGCATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTTCATCGGTA
TCATTACCCCCATGAACAGAAATCCCCCTTACACGGAGGCATCAGTGACCAAACAGGAAAAAACCGCCCTTAACATGGCC
CGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATGAACAGGCAGACATCTGTGA
ATCGCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACA
TGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGT
GTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCA
GAGCAGATTGTACTGAGAGTGCACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGG
CGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGC
GGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACC
GTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAG
AGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGAC
CCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATC
TCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCC
GGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAG
AGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTA
TCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGC
GGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGG
GTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAACAATAAAACTGTCTGCTTACATAAACAGT
AATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCTTGCTCTAGGCCGCGATTAAATTCCAACATGGATGCTGA
TTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATG
CGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTG
ACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCC
CGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGC
GCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGA
ATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAAT
GCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGG
GGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGC
CTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCA
GTTTCATTTGATGCTCGATGAGTTTTTCTAAGAATTAATTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAA
CAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTT
AAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCG
AGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAA
ACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACT
AAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGA
AAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAAT
GCGCCGCTACAGGGCGCGTCCCATTCGCCA
93
5. Appendix
5.1.3
Expression plasmids
5.1.3.1 T7 RNAP wildtype-pGDR11
Restriction sites: SphI and HindIII
Mutation Y639F: TAT → TTT
Mutation H784A: CAT → GCT
Mutation E593G: GAA → GGC
Mutation Y639V: TAT → GTT
Mutation V685A: GTT → GCG
Mutation H784G: CAT → GGT
I→ORF
…GAAATTAACTATGAGAGGATCTCACCATCACCATCACCATACGGATCCGCATGCAATTGAAGGTCGCATGAATACCATT
AATATTGCCAAAAATGATTTTAGCGATATTGAACTGGCAGCCATTCCGTTTAATACCCTGGCAGATCATTATGGTGAACG
TCTGGCACGTGAACAGCTGGCACTGGAACATGAAAGCTATGAAATGGGTGAAGCCCGTTTTCGTAAAATGTTTGAACGTC
AGCTGAAAGCCGGTGAAGTTGCAGATAATGCAGCAGCAAAACCGCTGATTACCACCCTGCTGCCGAAAATGATTGCCCGT
ATTAATGATTGGTTTGAAGAAGTGAAAGCCAAACGCGGTAAACGTCCGACCGCATTTCAGTTTCTGCAGGAAATTAAACC
GGAAGCCGTTGCCTATATTACCATTAAAACCACCCTGGCATGTCTGACCAGCGCAGATAATACCACCGTTCAGGCAGTTG
CAAGCGCAATTGGTCGTGCAATTGAAGATGAAGCACGCTTTGGTCGTATTCGTGATCTGGAAGCCAAACATTTTAAAAAA
AATGTGGAAGAACAGCTGAATAAACGTGTGGGCCATGTGTATAAAAAAGCCTTTATGCAGGTTGTTGAAGCAGATATGCT
GAGCAAAGGTCTGCTGGGTGGTGAAGCATGGTCTAGCTGGCATAAAGAAGATAGCATTCACGTTGGTGTTCGCTGTATTG
AAATGCTGATTGAAAGCACCGGTATGGTTAGCCTGCATCGTCAGAATGCCGGTGTTGTTGGTCAGGATAGCGAAACCATT
GAACTGGCTCCGGAATATGCAGAAGCAATTGCAACACGCGCAGGCGCACTGGCAGGTATTAGCCCGATGTTTCAGCCGTG
TGTTGTTCCTCCGAAACCGTGGACAGGTATTACAGGTGGTGGTTATTGGGCAAATGGTCGTCGTCCGCTGGCACTGGTTC
GTACCCATAGCAAAAAAGCCCTGATGCGTTATGAAGATGTGTATATGCCGGAAGTGTATAAAGCGATTAATATTGCGCAG
AATACCGCCTGGAAAATTAATAAAAAAGTGCTGGCCGTTGCAAATGTTATTACCAAATGGAAACATTGTCCGGTTGAAGA
TATTCCGGCAATTGAACGTGAAGAACTGCCGATGAAACCGGAAGATATTGATATGAATCCGGAAGCACTGACCGCATGGA
AACGTGCAGCAGCAGCAGTTTATCGTAAAGATAAAGCCCGTAAAAGCCGTCGTATTAGCCTGGAATTTATGCTGGAACAG
GCCAATAAATTTGCCAATCATAAAGCCATTTGGTTTCCGTATAATATGGATTGGCGTGGTCGTGTTTATGCAGTGAGCAT
GTTTAATCCGCAGGGTAATGATATGACCAAAGGCCTGCTGACCCTGGCAAAAGGTAAACCGATTGGCAAAGAAGGTTATT
ATTGGCTGAAAATTCATGGTGCAAATTGTGCCGGTGTTGATAAAGTTCCGTTTCCGGAACGCATTAAATTTATTGAAGAA
AACCATGAAAATATTATGGCCTGTGCAAAATCTCCGCTGGAAAATACCTGGTGGGCAGAACAGGATAGCCCGTTTTGTTT
TCTGGCCTTTTGCTTTGAATATGCCGGTGTTCAGCATCATGGTCTGAGCTATAATTGTAGCCTGCCGCTGGCATTTGATG
GTAGCTGTAGCGGTATTCAGCATTTTAGCGCAATGCTGCGTGATGAAGTTGGTGGTCGTGCAGTTAATCTGCTGCCGAGC
GAAACCGTTCAGGATATTTATGGCATTGTGGCCAAAAAAGTGAATGAAATTCTGCAGGCCGATGCAATTAATGGCACCGA
TAATGAAGTTGTTACCGTGACCGATGAAAACACCGGTGAAATTAGCGAAAAAGTGAAACTGGGCACCAAAGCCCTGGCTG
GTCAGTGGCTGGCATATGGTGTTACCCGTAGCGTTACCAAACGTAGCGTTATGACCCTGGCCTATGGTAGCAAAGAATTT
GGTTTTCGTCAGCAGGTTCTGGAAGATACCATTCAGCCTGCAATTGATAGCGGTAAAGGCCTGATGTTTACCCAGCCGAA
TCAGGCAGCAGGTTATATGGCAAAACTGATTTGGGAAAGCGTTAGCGTTACCGTTGTTGCAGCAGTTGAAGCAATGAATT
GGCTGAAATCTGCAGCAAAACTGCTGGCAGCAGAAGTGAAAGATAAAAAAACAGGCGAAATTCTGCGTAAACGTTGTGCA
GTTCATTGGGTTACACCGGATGGTTTTCCGGTTTGGCAGGAATATAAAAAACCGATCCAGACCCGTCTGAACCTGATGTT
TCTGGGTCAGTTTCGTCTGCAGCCGACCATTAATACCAATAAAGATAGCGAAATTGATGCCCATAAACAGGAAAGCGGTA
TTGCACCGAATTTTGTGCATAGCCAGGATGGTAGCCATCTGCGTAAAACCGTTGTTTGGGCACATGAAAAATATGGCATT
GAATCCTTTGCCCTGATTCATGATAGCTTTGGCACCATTCCGGCAGATGCAGCAAACCTGTTTAAAGCCGTGCGTGAAAC
CATGGTTGATACCTATGAAAGCTGTGATGTTCTGGCCGATTTTTATGATCAGTTTGCCGATCAGCTGCATGAAAGCCAGC
TGGATAAAATGCCTGCACTGCCTGCCAAAGGTAATCTGAATCTGCGCGATATTCTGGAAAGCGATTTTGCCTTTGCCTAA
AAGCTTAATT…
94
5. Appendix
5.1.3.2 KOD (exo-) wildtype-pET24a(+)
Restriction sites: NdeI and HindIII
Mutation L408Q: CTG → CAG
Mutation A485L: GCA → CTG
I→ORF
…AGATATACATATGATTCTGGATACCGATTACATTACCGAAGATGGTAAACCGGTGATTCGCATTTTTAAAAAAGAAAAC
GGCGAATTTAAAATCGAATATGATCGCACCTTTGAACCGTATTTTTATGCACTGCTGAAAGATGATAGCGCCATTGAAGA
AGTGAAAAAAATTACCGCAGAACGTCATGGCACCGTTGTTACCGTTAAACGTGTTGAAAAAGTGCAGAAAAAATTTCTGG
GTCGTCCGGTTGAAGTGTGGAAACTGTATTTTACCCATCCGCAGGATGTTCCGGCAATTCGTGATAAAATTCGTGAACAT
CCGGCAGTGATTGATATTTATGAATATGATATTCCGTTTGCCAAACGCTATCTGATTGATAAAGGTCTGGTTCCGATGGA
AGGTGATGAAGAACTGAAAATGCTGGCATTTGCAATCGCAACCCTGTATCATGAAGGTGAAGAATTTGCCGAAGGTCCGA
TTCTGATGATTAGCTATGCAGATGAAGAAGGTGCACGCGTTATTACCTGGAAAAATGTTGATCTGCCGTATGTTGATGTT
GTTAGCACCGAACGCGAAATGATTAAACGTTTTCTGCGTGTGGTGAAAGAAAAAGATCCGGATGTTCTGATTACCTATAA
TGGCGATAATTTTGATTTTGCCTATCTGAAAAAACGCTGCGAAAAACTGGGCATTAATTTTGCACTGGGTCGTGATGGTA
GCGAACCGAAAATTCAGCGTATGGGTGATCGTTTTGCCGTTGAAGTTAAAGGTCGCATTCATTTTGATCTGTATCCGGTT
ATTCGTCGCACCATTAATCTGCCGACCTATACCCTGGAAGCAGTTTATGAAGCAGTTTTTGGTCAGCCGAAAGAAAAAGT
TTATGCCGAAGAAATTACCACCGCATGGGAAACAGGCGAAAATCTGGAACGTGTTGCACGTTATAGCATGGAAGATGCAA
AAGTTACCTATGAACTGGGCAAAGAATTTCTGCCGATGGAAGCACAGCTGAGCCGTCTGATTGGTCAGAGCCTGTGGGAT
GTTAGCCGTAGCAGCACCGGTAATCTGGTTGAATGGTTTCTGCTGCGTAAAGCCTATGAACGTAATGAACTGGCACCGAA
TAAACCGGATGAAAAAGAACTGGCACGTCGTCGTCAGAGCTATGAAGGTGGTTATGTTAAAGAACCGGAACGTGGTCTGT
GGGAAAATATTGTTTATCTGGATTTTCGCAGCCTGTATCCGAGCATTATTATTACCCATAATGTGAGTCCGGATACCCTG
AATCGTGAAGGTTGTAAAGAATATGATGTTGCACCGCAGGTTGGTCATCGTTTTTGTAAAGATTTTCCGGGTTTTATTCC
GAGCCTGCTGGGTGATCTGCTGGAAGAACGTCAGAAAATTAAAAAAAAAATGAAAGCCACCATTGATCCGATTGAACGTA
AACTGCTGGATTATCGTCAGCGTGCAATTAAAATTCTGGCCAATAGTTATTATGGCTATTATGGTTATGCACGTGCCCGT
TGGTATTGTAAAGAATGTGCAGAAAGCGTTACCGCATGGGGTCGTGAATATATCACCATGACCATTAAAGAAATTGAAGA
AAAATACGGCTTTAAAGTGATTTATAGCGATACCGATGGCTTTTTTGCAACCATTCCGGGTGCAGATGCAGAAACCGTTA
AAAAAAAAGCCATGGAATTTCTGAAATATATTAATGCCAAACTGCCGGGTGCACTGGAACTGGAATATGAAGGTTTTTAT
AAACGCGGTTTTTTTGTGACCAAAAAAAAATATGCCGTGATTGATGAAGAAGGCAAAATTACCACCCGTGGTCTGGAAAT
TGTTCGTCGTGATTGGAGCGAAATTGCAAAAGAAACCCAGGCACGTGTTCTGGAAGCCCTGCTGAAAGACGGTGATGTTG
AAAAAGCCGTGCGCATTGTTAAAGAAGTTACCGAAAAACTGAGCAAATATGAAGTTCCGCCGGAAAAACTGGTGATTCAT
GAGCAGATTACCCGTGATCTGAAAGATTATAAAGCAACCGGTCCGCATGTTGCAGTTGCAAAACGTCTGGCAGCACGTGG
TGTTAAAATTCGTCCGGGTACAGTGATTAGCTATATTGTTCTGAAAGGTAGCGGTCGTATTGGTGATCGTGCAATTCCGT
TTGATGAATTTGATCCGACCAAACATAAATATGATGCCGAATATTATATTGAAAATCAGGTTCTGCCGGCTGTTGAACGT
ATTCTGCGTGCATTTGGTTATCGTAAAGAAGATCTGCGCTATCAGAAAACACGTCAGGTTGGTCTGAGCGCATGGCTGAA
ACCGAAAGGCACCTTACTCGAGGGCAGCGGCAGCACCACCGAAAACCTGTATTTTCAGGGCTCAAAGCTTGCGGCCGCAC
TCGAGCACCACCACCACCACCACTGAGATCCGGCTG…
5.1.4
Protein sequences
5.1.4.1 T7 RNA polymerase wildtype
MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPK
MIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAK
HFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQD
SETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAI
NIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEF
MLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIK
FIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVN
LLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYG
SKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILR
KRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHE
KYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDF
AFA
95
5. Appendix
5.1.4.2 T7 RNA polymerase mutant M1 (Y639F, H784A)
MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPK
MIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAK
HFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQD
SETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAI
NIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEF
MLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIK
FIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVN
LLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAFG
SKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILR
KRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVASQDGSHLRKTVVWAHE
KYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDF
AFA
5.1.4.3 T7 RNA polymerase mutant M2 (E593G, Y639V, V685A, H784G)
MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPK
MIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAK
HFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQD
SETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAI
NIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEF
MLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIK
FIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVN
LLPSETVQDIYGIVAKKVNEILQADAINGTDNGVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAVG
SKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESASVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILR
KRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVGSQDGSHLRKTVVWAHE
KYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDF
AFA
5.1.4.4 T7 RNA polymerase mutant 2P16 (I119V, G225S, K333N, D366N,
F400L, E593G, Y639V, S661G, V685A, H784, F880Y)
MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPK
MIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITVKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAK
HFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTSMVSLHRQNAGVVGQD
SETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAI
NIAQNTAWKINKNVLAVANVITKWKHCPVEDIPAIEREELPMKPENIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEL
MLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIK
FIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVN
LLPSETVQDIYGIVAKKVNEILQADAINGTDNGVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAVG
SKEFGFRQQVLEDTIQPAIDGGKGLMFTQPNQAAGYMAKLIWESASVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILR
KRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVGSQDGSHLRKTVVWAHE
KYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDY
AFA
96
5. Appendix
5.1.4.5 KOD exo- DNA polymerase wildtype
MILDTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYFYALLKDDSAIEEVKKITAERHGTVVTVKRVEKVQKKFLGRPV
EVWKLYFTHPQDVPAIRDKIREHPAVIDIYEYDIPFAKRYLIDKGLVPMEGDEELKMLAFAIATLYHEGEEFAEGPILMI
SYADEEGARVITWKNVDLPYVDVVSTEREMIKRFLRVVKEKDPDVLITYNGDNFDFAYLKKRCEKLGINFALGRDGSEPK
IQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGQPKEKVYAEEITTAWETGENLERVARYSMEDAKVTY
ELGKEFLPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRRQSYEGGYVKEPERGLWENI
VYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKKKMKATIDPIERKLLD
YRQRAIKILANSYYGYYGYARARWYCKECAESVTAWGREYITMTIKEIEEKYGFKVIYSDTDGFFATIPGADAETVKKKA
MEFLKYINAKLPGALELEYEGFYKRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGDVEKAV
RIVKEVTEKLSKYEVPPEKLVIHEQITRDLKDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEF
DPTKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLSAWLKPKGT
5.1.4.6 KOD exo- DNA polymerase mutant L408Q
MILDTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYFYALLKDDSAIEEVKKITAERHGTVVTVKRVEKVQKKFLGRPV
EVWKLYFTHPQDVPAIRDKIREHPAVIDIYEYDIPFAKRYLIDKGLVPMEGDEELKMLAFAIATLYHEGEEFAEGPILMI
SYADEEGARVITWKNVDLPYVDVVSTEREMIKRFLRVVKEKDPDVLITYNGDNFDFAYLKKRCEKLGINFALGRDGSEPK
IQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGQPKEKVYAEEITTAWETGENLERVARYSMEDAKVTY
ELGKEFLPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRRQSYEGGYVKEPERGLWENI
VYLDFRSQYPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKKKMKATIDPIERKLLD
YRQRAIKILANSYYGYYGYARARWYCKECAESVTAWGREYITMTIKEIEEKYGFKVIYSDTDGFFATIPGADAETVKKKA
MEFLKYINAKLPGALELEYEGFYKRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGDVEKAV
RIVKEVTEKLSKYEVPPEKLVIHEQITRDLKDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEF
DPTKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLSAWLKPKGT
5.1.4.7 KOD exo- DNA polymerase mutant L408Q, A485L
MILDTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYFYALLKDDSAIEEVKKITAERHGTVVTVKRVEKVQKKFLGRPV
EVWKLYFTHPQDVPAIRDKIREHPAVIDIYEYDIPFAKRYLIDKGLVPMEGDEELKMLAFAIATLYHEGEEFAEGPILMI
SYADEEGARVITWKNVDLPYVDVVSTEREMIKRFLRVVKEKDPDVLITYNGDNFDFAYLKKRCEKLGINFALGRDGSEPK
IQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGQPKEKVYAEEITTAWETGENLERVARYSMEDAKVTY
ELGKEFLPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRRQSYEGGYVKEPERGLWENI
VYLDFRSQYPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKKKMKATIDPIERKLLD
YRQRLIKILANSYYGYYGYARARWYCKECAESVTAWGREYITMTIKEIEEKYGFKVIYSDTDGFFATIPGADAETVKKKA
MEFLKYINAKLPGALELEYEGFYKRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGDVEKAV
RIVKEVTEKLSKYEVPPEKLVIHEQITRDLKDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEF
DPTKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLSAWLKPKGT
97
5. Appendix
5.2
Abbreviations
Amino acid nomenclature
A
Ala
Alanine
M
Met
Methionine
C
Cys
Cysteine
N
Asn
Asparagine
D
Asp
Aspartate
P
Pro
Proline
E
Glu
Glutamate
Q
Gln
Glutamine
F
Phe
Phenylalanine
R
Arg
Arginine
G
Gly
Glycine
S
Ser
Serine
H
His
Histidine
T
Thr
Threonine
I
Ile
Isoleucine
V
Val
Valine
K
Lys
Lysine
W
Trp
Tryptophane
L
Leu
Leucine
Y
Tyr
Tyrosine
32
P
Phosphorus isotope 32P
A
Adenine
APS
Ammonium persulfate
ATP
Adenosine 5’-triphosphate
bp
base pairs
BSA
Bovine serum albumin
C
Cytosine
CTP
Cytidine 5’-triphosphate
dATP
2’-Desoxyadenosine 5’-triphosphate
DEAE
Diethylaminoethyl
dCTP
2’-Desoxycytidine 5’-triphosphate
dGTP
2’-Desoxyguanosine 5’-triphosphate
DMF
Dimethylformamide
DMSO
Dimethyl sulfoxide
DNA
Deoxyribonucleic acid
dNTP
2’-Desoxyribonucleoside 5’-triphosphate
dsDNA
double-stranded DNA
DTT
1,4-Dithiotreitol
E. coli
Escherichia coli
EDTA
Ethylenediaminetetraacetic acid
ESI-MS
Electrospray mass spectrometry
98
5. Appendix
EtOH
Ethanol
exo-
3’-5’ Exonuclease deficient
FPLC
Fast protein liquid chromatography
G
Guanine
h
hours
HRMS
High resolution mass spectrometry
IMAC
Immobilized metal ion affinity chromatography
IPTG
Isopropyl-β-D-thiogalactopyranoside
kb
kilobases
kDa
kilodaltons
Klentaq
Klenow fragment of Taq DNA polymerase
KTQ
Klentaq
LB
Lysogeny Broth
M
Molar [mol/L]
min
minute(s)
MPLC
Medium pressure liquid chromatography
MWCO
Molecular Weight Cut-Off
Ni-IDA
Nickel iminodiacetic acid
NMR
Nuclear magnetic resonance
nt
nukleotide(s)
NTP
Ribonucleoside 5’-triphosphate
OD600
Optical density (λ = 600 nm)
PAGE
Polyacrylamide gel electrophoresis
PCR
Polymerase chain reaction
PMSF
Phenylmethylsulfonyl fluoride
RNA
Ribonucleic acid
RP
Reversed phase
rpm
Rotations per minute
s
second(s)
SDS
Sodium dodecyl sulfate
SELEX
Systematic Evolution of Ligands by Exponential Enrichment
ssDNA
single stranded DNA
t
time
T
Thymine
TAE
Tris-acetate-EDTA
Taq
Thermus aquaticus
TBE
Tris-borate-EDTA
99
5. Appendix
TEAA
Triethylammonium acetate
TEAB
Triethylammonium bicarbonate
TEMED
N, N, N’, N’-Tetramethylethylenediamine
TLC
Thin layer chromatography
TMP
Trimethyl phosphate
TMS
Trimethylsilane
Tris
Tris(hydroxymethyl)aminomethane
TTP
Thymidine 5’-triphosphate
U
Uracil, units
UTP
Uridine 5’-triphosphate
WT
Wildtype
UV
Ultraviolet
100
6. References
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