Discrete Assembly of Synthetic Peptide-DNA Triplex Structures from Polyvalent
Melamine-Thymine Bifacial Recognition
THESIS
Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in
the Graduate School of The Ohio State University
By
Yingying Zeng, B.S.
Graduate Program in Chemistry
The Ohio State University
2013
Master's Examination Committee:
Dr. Claudia Turro, Advisor
Dr. Jon Parquette
Copyright by
Yingying Zeng
2013
Abstract
Triplex-forming oligonucleotides (TFOs) and peptide nucleic acids (PNAs) haven been
known to direct DNA triplex assembly via Hoogsteen or reverse Hoogsteen base-pairing.
However, with this conventional approach, target DNA sequences are almost solely
homopurines. While the Janus-Wedge targeting concept has also been an elegant
expansion of the recognition code for DNA triplex formation, it was largely restricted to
preformed duplex assembly. There are fewer synthetic systems to induce triplex
structures in single stranded oligonucleotides. With these goals in mind, we have
designed and synthesized a 21-residue α-peptide (EM*)10G that simultaneously
recognizes two oligodeoxythymidine (dT10) tracts to form triplexes with a peptide-DNA
strand ratio of 1:2. The synthetic peptide has a controlled display of 10 melamine rings on
lysine side chains (termed as M*), which addresses bifacial thymine-recognition
interfaces along the length of the 21-residue peptide. Alternate residues are glutamic acid
bearing negative charges to afford aqueous solubility and avoid nonspecific electrostatic
binding with DNA. Binding stoichiometry was obtained by UV and fluorescence
titration, indicating the formation of ternary peptide•[dT10]2 complex as well as
heterodimeric peptide•[dT10C10T10] hairpin structure with triplex stems. Signal change on
circular dichroism (CD) provided the direct evidence of DNA conformational change
ii
induced by the synthetic peptide. Clean transition trend from DNA to DNA-peptide
complex bands based on native electrophoresis further supported discrete peptide-DNA
assembly. Monophasic transition with cooperative melting was observed for both triplex
and hairpin by UV and fluorescence denaturation. Highly exothermic assembly profiles (28.5 kcal/mol for triplex and -31.4 kcal/mol for hairpin per peptide-DNA triplet stack)
from differential scanning calorimetry (DSC) were comparable to those of DNA-DNA
and melamine-cyanuric acid recognition, suggestive of similar driving forces. Binding
affinity was quantified using fluorescence quenching and fluorescence anisotropy,
yielding apparent dissociation constant (Kd) 4000 nM2 for the triplex and 2.7 nM for the
hairpin. Recognition exhibited selectivity for thymine over other nucleobases. Further
more, partial and full methylation of melamine rings on the peptide abolished all
detectable binding to dT10 tract, supporting the idea that assembly depends on the
hydrogen-bonding
directed
melamine-thymine
bifacial
recognition
rather
than
nonspecific aggregation. We anticipate that this novel assembly element may present
promising artificial regulator to manipulate the structure and function of thymine- oruracil rich targets.
iii
Dedication
To my family
iv
Acknowledgments
As I reflect on my graduate study here at OSU, I am left in awe of how quickly time has
been passed and also glad to see myself growing toward an independent researcher and
mature individual. I would like to take this opportunity to acknowledge the great people
who have made this journey a worthwhile experience.
I owe a great debt of gratitude to my current research advisor, Profeesor Claudia Turro,
who took me in when I thought I could never see the program through. Her mentorship
and advice have motivated me through the tough times. I highly appreciate the
opportunity that she gave me to finish my master’s examination. I can nerver thank her
enough for her support and guidance. I would also like to thank the member of my thesis
committee, Professor Jon Parquette for offering invaluable advice and support.
I am also gratedful to my former research advisor, Professor Dennis Bong, for giving me
such a great research opportunity to work on the project presented in this thesis. I highly
treasure the time being part of his research team. Through those inspiring discussions
with him, I have learned the rational and critical way of thinking when approaching a
scientific problem. It will be invaluable towards my future career development.
Throughout the years in the Bong lab, I’ve had the tremendou fortune to work with a
group of brilliant and motivated researchers. I would like to thank them all for everything
that I have learned. Particularly, among the former graduate students, I am glad to have
v
known Mingming Ma and Oscar Torres, who are great scientists and friends. I am very
grateful for their helpful discussions and advice on my academic study here.
In addition, I would like show my appreciation to the incredibly supportive staff here in
the chemistry department.
Finally and mostly, my greatest debt of gratitude is to my family, for their faith, love and
support in my Scientific Odyssey.
vi
I am a part of all that I have met.
Yet all experience is an arch where-thro’
Gleams that untravell’d world, whose margin fades
Foreever and forever when I move.
— From ULYSSES, Alfred, Lord Tennyson
vii
Vita
2002-2005……………………………………………Rui’an the 4th Middle School, China
2005-2009…………………………B.S., Chemistry, Sichuan University, Chengdu, China
2009-2013………………..................Graduate Teaching/ Research Assistant, Department
of Chemistry, The Ohio State University
Publications
1.
Zeng, Y.; Pratumyot, Y; Piao, X.; Bong, D. Discrete Assembly of Synthetic PeptideDNA Triplex Structures from Polyvalent Melamine-Thymine Bifacial Recognition.
J. Am. Chem. Soc., 2012, 134, 832-835.
Fields of Study
Major Field: Chemistry
viii
Table of Contents
Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita................................................................................................................................... viii List of Tables .................................................................................................................... xii List of Figures .................................................................................................................. xiii Chapter1. Introduction to Sequence-specific DNA Recognition by Triplex-Forming
Molecules (TFMs) .............................................................................................................. 1 1.1. Background and Significance ...................................................................................... 2 1.2. Structural Features of DNA ......................................................................................... 3 1.3. Strategies to Construct DNA Triplex ........................................................................... 5 1.3.1. Hoogsteen or Reverse Hoogsteen Approach ......................................................... 5 1.3.1.1. Nature’s Solution: Triplex-forming oligonucleotides (TFOs)........................ 5 1.3.1.2. Structural Features .......................................................................................... 8 1.3.1.3. TFOs Modifications...................................................................................... 10 ix
1.3.1.4. Triplex-forming Peptide Nucleic Acids (PNAs) .......................................... 15 1.3.2. Janus-Wedge Approach....................................................................................... 22 Chapter 2. Experimental: Design, Synthesis and Characterization .................................. 28 2.1. DNA Triplex-forming Peptide Design....................................................................... 29 2.2. Materials and Methods ............................................................................................... 32 2.2.1. General ................................................................................................................ 32 2.2.2. Synthetic schemes for monomer preparation ...................................................... 33 2.2.3. Synthetic procedures ........................................................................................... 34 2.2.4. Solid phase peptide synthesis and characterization............................................. 39 2.2.5. General sample preparation protocol for binding studies ................................... 45 2.2.6. UV Job Plot Experiments .................................................................................... 45 2.2.7. Fluorescence Job Plot (Quenching) Experiments ............................................... 45 2.2.8. Circular Dichroism (CD) Experiments ............................................................... 46 2.2.9. Thermal Denaturation Studies by UV ................................................................. 46 2.2.10. Thermal Denaturation Studies by Fluorescence................................................ 47 2.2.11. Differential Scanning Calorimetry (DSC)......................................................... 47 2.2.12. Fluorescence and Fluorescence Anisotropy ...................................................... 49 x
2.2.13. Gel Mobility Shift Assay .................................................................................. 51 Chapter 3. Results and Discussion .................................................................................... 53 3.1. Binding Stoichiometry ............................................................................................... 54 3.2. CD and Gel Mobility Shift......................................................................................... 56 3.3. Thermal Stability ....................................................................................................... 58 3.4. Binding Affinity ......................................................................................................... 60 3.5. Binding Selectivity..................................................................................................... 61 Chapter 4. Conclusion....................................................................................................... 66 Reference .......................................................................................................................... 69 Appendix A: Additional Characterization Data ................................................................ 77 Appendix B: 1H and 13C NMR Spectra of Amino Acid Derivatives ................................ 81 xi
List of Tables
Table 1.1. Thermal Stabilities of PNA-DNA triplexes. ................................................... 17 Table 1.2. Thermal stabilities of J-W triplexes. ............................................................... 26 xii
List of Figures
Figure 1.1. Structural features of DNA double helix. (A) The sugar-phosphate backbones
of complementary nucleotide strands are shown in red and orange, and the Watson-Crick
base pairs are shown in gray. (B) The chemical structures and electro-features of DNA
bases thymine (T), adenine (A), cytosine (C), and guanine (G) are shown as hydrogenbonded base pairs. ............................................................................................................... 3 Figure 1.2. Oligonucleotide-directed cleavage of double helical DNA by a triplexforming DNA-EDTA•Fe probe. ......................................................................................... 6 Figure 1.3. (Left) Simplified model of the triple helix complex between a Hoogsteen
bound DNA-EDTA•Fe probe at single site within 4.06 kb of plasmid DNA. (Right)
Isomorphous base triplets of TAT and C+GC. ................................................................... 7 Figure 1.4. Hydrogen bonding pattern of base triplets formed by natural nucleobases
through either Hoogsteen (upper left) or reverse Hoogsteen (lower). ................................ 8 Figure 1.5. Classification of binding configurations of TFOs according to their
compositions. ...................................................................................................................... 9 Figure 1.6. Base triplets of C+-G-C and P1-G-C. ............................................................ 11 Figure 1.7. Hydrogen bonding pattern of m5oxC-G-C base triplet. .................................. 11 Figure 1.8. Hoogsteen hydrogen bonding of antiTA, antiAT, antiCG and antiGC Cglycosides to their Watson-Crick base pair partners......................................................... 13 xiii
Figure 1.9. Design of rationale for recognition of Watson-Crick CG base pair by
nonnatural bases D2 and D3 within a pyrimidine-purine-pyrimidine triplex motif. ......... 13 Figure 1.10. Representative structure of the 3’, 5’-NDI bisconjugate with NDI
intercalators attached to both the 3’ and 5’ end of an oligonucleotide. ............................ 14 Figure 1.11. (Upper) A DNA-naphthalene diimide conjugate; (Lower) A DNA-perylene
diimide conjugate. ............................................................................................................. 15 Figure 1.12. Generic chemical structure of PNAs (B = thyminyl) studied...................... 16 Figure 1.13. Schematic representation of strand displacement PNA binding mode upon
targeting double stranded DNA. ....................................................................................... 18 Figure 1.14. Triplex-forming peptide nucleic acid (PNA)............................................... 19 Figure 1.15. The studied PNA and DNA sequences. ....................................................... 20 Figure 1.16. Diagram of the bis-PNA-DNA triplex......................................................... 21 Figure 1.17. Lehn’s Janus-Wedge concept: Janus wedges with two hydrogen bonding
faces (A: acceptor, D: donor) are designed to bind by insertion between base-pairs
forming a triplet with the maximum number of Watson-Crick interactions. ................... 23 Figure 1.18. Lehn’s Janus-Wedge molecule capable of forming a triad with thymine and
cytosine derivatives in CHCl3. .......................................................................................... 23 Figure 1.19. (a) A Janus-Wedge base triplet: the third strand residue W binds to the
Watson-Crick faces of both target residues; (b) the 11dC8-11/11dT811 DNA target
sequence. ........................................................................................................................... 25 xiv
Figure 1.20. Janus-Wedge base triplets: (a) the third-strand residue W1 binds to the W-C
faces of target A and T; (b) W2 binds to G and C similarly.............................................. 25 Figure 2.1. Bifacial melamine-thymine recognition. ....................................................... 30 Figure 2.2. Synthesis of monomer 1. ............................................................................... 33 Figure 2.3. Synthesis of monomer 3. ............................................................................... 33 Figure 2.4. Synthesis of monomer 4. ............................................................................... 34 Figure 2.5. (A) MALDI-TOF of peptide 1; (B) HPLC trace of of peptide 1 on a C-18
analytical column using a gradient of 10-100% solvent B over 50 min, monitored by a
UV-Vis detector at 230 nm. .............................................................................................. 40 Figure 2.6. (A) MALDI-TOF of peptide 2; (B) HPLC trace of of peptide 2 on a C-18
analytical column using a gradient of 10-100% solvent B over 50 min, monitored by a
UV-Vis detector at 230 nm. .............................................................................................. 41 Figure 2.7. (A) MALDI-TOF of peptide 3; (B) HPLC trace of of peptide 3 on a C-18
analytical column using a gradient of 10-100% solvent B over 50 min, monitored by a
UV-Vis detector at 230 nm. .............................................................................................. 42 Figure 2.8. (A) MALDI-TOF of 5(6)-Carboxyfluorescein (Cbf) labeled peptide 3; (B)
HPLC trace of of Cbf labeled peptide 3 on a C-18 analytical column using a gradient of
10-100% solvent B over 50 min, monitored by a UV-Vis detector at 230 nm................. 43 xv
Figure 2.9. (A) MALDI-TOF of peptide 4; (B) HPLC trace of of peptide 4 on a C-18
analytical column using a gradient of 10-100% solvent B over 45 min, monitored by a
UV-Vis detector at 230 nm. .............................................................................................. 44 Figure 2.10. DSC traces of triplex 5 (black) with Non-2-State binding model fitted
curves shown in red. (A) Upscan and (B) Downscan. ...................................................... 48 Figure 2.11. DSC traces of Hairpin 6 (black) with Non-2-State binding model fitted
curves shown in red. (A) Upscan and (B) Downscan. ...................................................... 49 Figure 2.12. Representative fitting data set for triplex 5 ................................................. 50 Figure 2.13. Representative fitting data set for hairpin 6................................................. 51 Figure 3.1. Peptide 1 titrated into (A) dT10 and (B) Flc-dT10C10T10-Dabcyl, followed by
UV absorbance (260 nm) and fluorescein emission (521 nm), respectively. ................... 54 Figure 3.2. UV absorbance (260nm) of peptide 1 alone at different concentrations. ...... 55 Figure 3.3. Peptide 1 titrated into dT10C10T10 followed by UV absorbance (260 nm). ... 56 Figure 3.4. Circular dichroism spectra in DPBS, pH 7.4, of (A) Peptide 1 complexed
with dT10 ( ) vs dT10 alone (--) and (B) Peptide 1 complexed with dT10C10T10 () vs
dT10C10T10 alone(--). Peptide 1 in both is at 5µM concentration (—), while dT10 and
dT10C10T10 are maintained at 10 and 5 µM, respectively. Electrophoretic mobility shift
assays imaged by Cy5 fluorescence for (C) Cy5-dT10 (DNA1) and (D) Cy5-dT10C10T10
(DNA2) at 20 nM in each lane, with increasing peptide 1 concentration from left to right.
xvi
(E) Relative electrophoretic mobilities of the free DNA oligos and their peptide
complexes, a mixture of complex 5 and DNA1 shown in the central lane. ...................... 57
Figure 3.5. (A) First-derivative plot of melting transitions of triplex 5 (--) and hairpin 6
(—) followed by UV absorbance (260 nm). Normalized absorbance change is shown
inset. (B) DSC upscan traces of triplex 5 (—) and hairpin 6 (—), with downscan traces
shown as dashed regular and bold lines. ........................................................................... 58 Figure 3.6. (A) Fluorescence melting curves for hairpin 6 (—) and free Flc-dT10C10T10Dabcyl (--); (B) Corresponding first derivative curves for hairpin 6................................ 59 Figure 3.7. Binding isotherms in DPBS, pH 7.4, of (A) peptide 2 binding to dT10
followed by fluorescein quenching upon binding and (B) peptide 2 binding to dT10C10T10
followed by fluorescence anisotropy. Solid lines show fits to (A) trimer-monomer 1:2
binding model ([fraction bound peptide 2] = [DNA]2/Kd + [DNA]2, R2 ≥ 0.96) and (B)
1:1 binding model (corrected for fluorescence quenching): [Bound peptide 2] =
((([RT]+[DNAT]+Kd)-(sqrt((([RT]+[DNAT]+Kd)^2)-(4*[RT]*[DNAT]))))/2), where [RT] is
the total concentration of peptide 2 (25 nM), [DNAT] is the total concentration of
dT10C10T10 used in each binding reaction. R2 > 0.98. ....................................................... 61 Figure 3.8. (A) Absorbance (260 nm) vs Temperature plots for control peptide 3 and
peptide 4 with dT10. Concentrations: 2 µM peptide with 2 µM dT10. (B) Electrophoretic
mobility shift assay imaged by Cy5 fluorescence for Cy5-dT10 at 150 nM with increasing
concentrations of peptide 3 (0, 30, 50, 100, 150, 225, 300, 750, 1200, 1500 nM) from left
xvii
to right. Study for peptide 4 and Cy5-dT10 under the same condition yielded the same
image. ................................................................................................................................ 63 Figure 3.9. (A) Absorbance (260 nm) vs Temperature plots for peptide 1 with control
oligos dA10 and dC10. Absence of UV shift or melting signatures. Concentrations: 1 µM
peptide 1 with 2 µM dA10 or dC10. (B) Fluorescence () and Anisotropy (Δ) assays for
peptide 2 with control oligos dG5A10 and dC10. Peptide 2 concentration is constant at 25
nM. (C) Circular dichroism spectra for peptide 1 with control oligos dA10 and dC10.
Concentrations: 5 µM peptide 1 with 10 µM dA10 or dC10. .............................................. 64 Figure A1. Additional UV Job plot analysis of peptide 1 with dT10. .............................. 78 Figure A2. Additional CD spectrum: Titration of 10 µM dT10 with increasing
concentration of peptide 1. ................................................................................................ 78 Figure A3. Additional CD spectrum: free dC5T10C6/dG6T10G5 (T10 tracts 10 µM) and
their peptide 1 complexes. ................................................................................................ 79 Figure A4. Additional thermal denaturation curve for peptide 1 alone at different
concentrations. Absence of UV melting signature rules out possible secondary structures
from peptide itself. ............................................................................................................ 79 Figure A5. Additional stoichiometric binding curves for triplex 5 or hairpin 6 by titrating
500 nM peptide 2 with dT10 or dT10C10T10. Both fluorescence (—) and anisotropy ()
were monitored for triplex 5. Only anisotropy was recorded for hairpin 6 (Δ). ............... 80 xviii
Figure A6. Additional control Fluorescence () and Anisotropy (Δ) assays for free Cbf
in solution with (A) dT10 and (B) dT10C10T10 to rule out non-specific binding between the
fluorophore and DNA oligomers. ..................................................................................... 80 Figure B1. 1H NMR of 1a. ............................................................................................... 82 Figure B2. 1H NMR of 1. ................................................................................................. 82 Figure B3. 1H NMR of 3a. ............................................................................................... 83 Figure B4. 1H NMR of 3b................................................................................................ 83 Figure B5. 1H NMR of 3c. ............................................................................................... 84 Figure B6. 1H NMR of 3. ................................................................................................. 84 Figure B7. 1H NMR of 4a. ............................................................................................... 85 Figure B8. 1H NMR of 4b................................................................................................ 85 Figure B9. 1H NMR of 4. ................................................................................................. 86 Figure B10. 13C NMR of 1a. ............................................................................................ 86 Figure B11. 13C NMR of 1. .............................................................................................. 87 Figure B12. 13C NMR of 3a. ............................................................................................ 87 Figure B13. 13C NMR of 3b. ........................................................................................... 88 Figure B14. 13C NMR of 3c. ............................................................................................ 88 Figure B15. 13C NMR of 3. .............................................................................................. 89 Figure B16. 13C NMR of 4a. ............................................................................................ 89 Figure B17. 13C NMR of 4b. ........................................................................................... 90 xix
Figure B18. 13C NMR of 4. .............................................................................................. 90 xx
Chapter 1
Introduction to Sequence-specific DNA
Recognition by Triplex-Forming Molecules
(TFMs)
1
1.1. Background and Significance
Nucleic acids play the most crucial role in the development of biodiversity on earth, the
beginning of central dogma of molecular biology. Particularly, the DNA sequence
(genome) carries the digital information to encode the logic of life, responsible for
genetic information storage, heredity, expression and other metabolic functions.
Moreover, certain DNA sequences are known to be toxic and associated with diseases.
Therefore, the ability to specifically manipulate DNA information processing would offer
a wide range of applications in biology, gene-based biotechnology and eventually genebased therapeutics.1 One way to approach this goal would be the rational design of
artificial ligands that can recognize and assemble to desired DNA sequences, thus can
provide powerful tools to interfere with genetic information flow. Synthetic molecules
can be made to bind to DNA with high sequence specificity and affinity when they are
instructed to do so. The instructions exist in the form of the molecule’s shape, its
chemical interfaces, and how well it fills the space where the recognition and assembly
events take place which are all required to be ‘complementary’ to the targeted DNA
fragment. These instructions are written into the molecule during its synthesis.2
2
1.2. Structural Features of DNA
The importance of the DNA double helix with all those functions originates from its
unique features of chemical structure and conformation. The biological relevant B-form
DNA is naturally assembled in a double helix structure, which was discovered by Watson
and Crick in 1953.3 Two complementary, antiparallel polydeoxyribonucleotide strands
consist of highly negatively charged sugar-phosphate backbones and more importantly
the stacked base pairs via specific hydrogen bonding. The chemical ‘signatures’ of the
nucleobases are characterized by the pattern of functional groups exposed at their
molecular surface (Figure 1.1). The hydrogen-bonding donor and acceptor arrays of the
nucleobases presented by a given DNA sequence provide the basis for molecular
3
recognition by naturally occurring or synthetic DNA ligands.
strands defines the helical grooves, within which the edges of the heterocyclic bases
are exposed (Figure 1.1A). The biologically relevant B-form structure of the DNA
continued
Figure 1.1. Structural features of DNA double helix. (A) The sugar-phosphate backbones of
complementary nucleotide strands are shown in red and orange, and the Watson-Crick base pairs
are shown in gray. (B) The chemical structures and electro-features of DNA bases thymine (T),
adenine (A), cytosine (C), and guanine (G) are shown as hydrogen-bonded base pairs. Adapted
from reference 4.
3
Figure 1.1 continued
B
Major Groove
Major Groove
Minor Groove
Minor Groove
Major Groove
Major Groove
Minor Groove
Minor Groove
More interestingly, DNA is polymorphic macromolecules that can adopt a variety of
Figure 1.1 Structural features of the DNA double helix. (A) Molecular rendering of B-form DNA
conformations
deviatebackbones
from B-form
duplex structure,
suchstrands
as single-,
triple-,in multidouble
helix. Sugar that
phosphate
of complementary
nucleotide
are shown
red
and orange. (B) Chemical features and electropotential surfaces of Watson-Crick base pairings.
5,6
Figure
1.1 complex
Structural as
features
DNAunusual
double helix.
(A) Molecular
rendering
B-form
DNA
stranded
well of
as the
other
structures,
which
may inof turn
reflect
double helix. Sugar phosphate backbones of complementary nucleotide strands are shown in red
and orange. (B) Chemical features and electropotential surfaces of Watson-Crick base pairings.
important signals during DNA replication as well as gene regulation, recombination and
mutation.7 Therefore, the formation of non-B-form duplex structures could potentially
present great opportunities to probe and modulate genetic information processing. In
particular, among related scientific efforts, DNA triplex structures including design
strategies and potential applications have been under intensive investigation.
4
1.3. Strategies to Construct DNA Triplex
There are two major classes of DNA triplex-forming molecules (TFMs) according to the
targeting concept: (1) via Hoogsteen or reverse Hoogsteen base pairing; (2) via JanusWedge base pairing.
1.3.1. Hoogsteen or Reverse Hoogsteen Approach
1.3.1.1. Nature’s Solution: Triplex-forming oligonucleotides (TFOs)
Intermolecular triplexes can be formed between triplex-forming oligonucleotides (TFOs)
and the target sequences on duplex DNA.8 The very first example of nucleic acids triple
helix structure was found in synthetic polyribonucleotides in 1957.9 OD titration and
ultracentrifuge indicated a stable complex involving two poly(U) strands for each
poly(A) strand in the presence of 10mM MgCl2. Besides, this sort of association behavior
exhibited a fair amount of specificity as the (A+U) didn’t pick up poly(C), poly(I) or
poly(A) as the third strand but only poly(U). During the three decades that followed this
initial discovery, several other polynucleotides triplex structures were reported.10,11
Poly(C) could also form a triplex with poly(G) at low pH.12-14 More importantly, the Xray
diffraction
patterns
of
triple-stranded
fibers
poly(A)•2poly(U)
and
poly(dA)•2poly(dT) suggested an A-RNA-like conformation of the two Watson-Crick
base-paired strands with the third strand in a parallel orientation bound to the homopurine
strand of the duplex by Hoogsteen hydrogen bonds.15
It was not demonstrated until 1987 that an oligonucleotide can bind sequence-specifically
to an oligopurine-oligopyrimidine duplex tract in the major groove.16,17 Dervan and co5
workers designed a series of homopyrimidine oligonucleotides with EDTA•Fe covalently
attached at a singe position that can specifically bind to homopurine-homopyrimidine
stranded complex (18, 19). Several investigators proposed an anti-
(DNA-EDTA) which cleave the com
orientation
thetriplex
two polypyrimidine
on the basis
of quence
parallel
A homopyrimidine ol
(26, 27).1.2
tracts within a large
DNA
duplexofby
formationstrands
and cleave
at that
site (Figure
an anti conformation of the bases (17-19). The x-ray diffraction a DNA cleaving moiety should recog
patterns of triple-stranded fibers (poly(A)-2 poly(U) and poly(dA)-2 plementary sequence of double helic
and 1.3). The specific
location
and this
asymmetry
the cleavage
pattern
a definite
an A'provided
and suggested
poly(dT))
supported
hypothesisof
(20-22)
and yield a strand break at
dine DNA
RNA-like conformation of the two Watson-Crick base-paired The affinity cleaving method (5, 26, 2
in thebind
strands with the third strand
samein
conformation,
effect oftoreaction
proof that the homopyrimidine-EDTA
probes
the major bound
groovethe
parallel
the conditions, pr
parallel to the homopurine strand of the duplex by Hoogsteen
mismatches on triple helix formatio
hydrogen bonds (23). The 12-fold helix with dislocation of the axis resolution sequencing gels. In addition
homopurine strandbyofalmost
Watson-Crick
double
helical
DNA.
oligonucleotides
in as
this
3 A, the C3'-endo
sugar
puckering
and Such
small base-tilts
strand as well
the identity of the gr
result in a large and deep major groove that is capable of accommo- helix occupied by the bound DNA-EDT
a high-resolution gel electrophoresis (5
strand
the thirdto
dating
(24). A high-resolution
x-ray structure of
context are nowadays
referred
as triplex-forming
oligonucleotides
(TFOs).
triple helical DNA or RNA has not yet been reported.
location of triple helices within large p
Oligonucleotide-EDTA
probes. Oligonucleotides equipped by double strand breaks analyzed by
with a DNA cleaving moiety have been described which produce electrophoresis. Nine homopyrimidin
sequence-specific cleavage of single-stranded DNA (25-28). An nucleotides in length, containing a si
example of this is oligonucleotide-EDTAFe hybridization probes covalently attached at the 5 position (la
6B) were synthesized for binding an
different duplex target DNA's. We find
3'
5'
EDTA probes bind the correspondin
3'
5'
duplex DNA in parallel orientation an
3'
5'
and dithiothreitol (DTT), cleave on
Watson-Crick DNA at that site.
One convenient synthesis of DNAI7T
incorporation of a modified thymidin
chemical methods. This approach all
3'
5'
and affords control over the precise loc
5'
3'3'
any thymidine position in the oligonuc
cleotides-EDTA 1 to 9 of differen
EDTA-thymidine position were synthe
the nine DNA-EDTA probes was pur
OH
02
Orientation and groove location of
Fig. 2. Oligonucleotide-directed cleavage of double helical DNA by a triple
helix-forming DNA-EDTA Fe probe. One thymidine has been replaced by Watson-Crick DNA. Although it is
Figure 1.2. Oligonucleotide-directed
of double
helical
DNA
byReduca triplex-forming
DNA-in triple helical DN
pyrimidine strands
attached
at C-5.
chelator EDTA
covalently
thymidine with the ironcleavage
tion of dioxygen generates localized hydroxyl radical at this position (26).
definite proof is lacking. We examined
EDTA•Fe probe. One thymine has been replaced by thymine with the iron chelator EDTA
covalently attachedFig.
at C-5.
Reduction of oxygen generates localized hydroxyl radical at this
3. (A) Autoradiogram of the 20 percent A
Maxam-Gilbert sequencing gel. (Lanes 1 to 5) 5'
10) 5' endlabeled d(CIOA15T5). (Lanes 1 and 6) The
Maxam-Gilbert G+A sequencing reactions (38,
39). (Lanes 2 and 7) Controls showing the two 5'
labeled 30-bp DNA standards obtained by treatment according to the cleavage reactions in the
absence of DNA-EDTAFe probes. (Lanes 3 to 5
and 8 to 10) The DNA cleavage products in the
presence of DNA-EDTA Fe probes 1 to 3, -0.5
1M (bp) 5' 32P-labeled DNA, (-10,000 count/
min), 10mMtris-HCl,pH 7.4, 1rmMspermine,
100 mM NaCI, 100 pM (bp) sonicated,depro-
position. Adapted from
reference
16. (lanes 6 to
end-labeled
d(A5T15G10);
1 2 3 4 5
6 7 8 9 10
B5'
-
5
S'-*TT
5'-
TT
'
DNA-EDTA
..3'-
1
GGGGGGGGGG
teinizedcalf thymus DNA, 40 percent (by volume) ethylene glycol, 0.67 PM probe, 25 PM
Fe(II), and 1 mM DIT; 15 hours, 0?C. (Lanes 3
and 8) DNA-EDTA Fe 1; (lanes 4 and 9) DNAEDTA Fe 2; (lanes 5 and 10) DNA-EDTA Fe 3.
6 T* is the
(B) (T)15-EDTA probes 1 to 3 where
positionof the thymidine-EDTA.Histogramsof
the DNA cleavage patterns derivedby densitometry of the autoradiogram shown in (A) (lanes 3 to
5 and 8 to 10). The heights of the arrows
represent the relative cleavage intensities at the
indicated bases. Arrows are shown if the cleavage
intensity at a particular nucleotide was greater
than 5 percent compared to the nucleotide cleaved
the most efficiently. The box indicates the double,
strandedsequence which is bound by the DNA-
9
_
NADA
5- CCCCCCCCCC
3- GGGGGGGGGGI|T
DNA-EDTA
3
CCCCCCCCCC[XAAAAAA
HE SEQUENCE-SPECIFIC
OF DOUBLE
corresponding
homopyrimidine
strandprobes
(17-19)
CLEAVAGE
HELICAL
(Fig.a1).
The DNA
to form
triple
for DNA-EDTAFe(II)
size dependence
DNA by restriction endonucleases is essential for many duplex
DNA-EDTA
Probes with
with the Watson-Crick DNA.
poly(dT-dC)poly(dG-dA)
associated
helix complex
poly(U-C) or
-wo0
in size,toproduce
11 nucleotides
13 the
and presence
6, which are
Fe(II) 5 and belowpH
6 in
of MgCl2
techniques in molecular biology,
including gene isolation, poly(dT-dC)
afford a triplecleavage patterns of similar intensities at 00C, indicating that
DNA sequence determination, and recombinant DNA manipulahomopurinehomopyrimidine sequences as short as 11 nucleotides
tions (1, 2). With the advent of pulsed-field gel electrophoresis, the
can specifically bind the 628-bp restriction fragment. The influence
separation of large pieces of DNA is now possible (3, 4). However,
of oligonucleotide length becomes more apparent
Major if the cleavage
Major
the binding site sizes of naturally occurring restriction enzymes are
groove
The DNAgroove
reactions are allowed to proceed at higher temperatures.
EDTA probes 4 and 5 still cleave the target duplex DNA at 250C
in the range of 4 to 8 base pairs, and hence their sequence
3'
5'
3' 5'
with approximately the same efficiency, whereas the relative intensispecificities may be inadequate for mapping genomes over very large
ty of the cleavage pattern
H
CH3 produced by the shorter 6 becomes
distances. The design of sequence-specific DNA cleaving molecules
significantly weaker
0 (Figs. 4A and 5B).
I
T-A- a
that go beyond the
of
the
natural
on
specificities
enzymes
depends
HHto test the importance
of sequence similarity
H for triple
In order
CL
~ ~ ~ ~ ~ ~ ~ ~ ~ TA-T
-C/
T-A
N
o
i
/
a
~~~~~~~3'
Ncleavage,
?
detailed understanding
of
the
chemical principles
underlying
two
we synthesized two probes, DNA-N
and,N
helix formation
-G-C
?H'
R
H
Y1
EDTAFe(II) 7 and 8, which contained single base mismatches
finctions: recognition and cleavage of DNA (5). Synthetic seN
N
N
location
common
the
4
had
in
but
to
DNA-EDTAFe(II)
compared
N
N
quence-specific binding moieties for double helical
DNA that have
*
~~~T-A-T
of T* at position 5. When bound to the double helical target
been studied are coupled analogs of natural products (5), transition
C-G- C
R mismatched
R
H base
probes 7 and 8 should give rise to one
sequence,
T-A-T
metal complexes (6), and peptide fragments derived from DNA
C+GC
triplet with respect to the Hoogsteen hydrogen bonding. The
C-LG-C
binding proteins (7, 8).
mismatching bases in the probe strands were chosen so that the
T-A-T
protonated structures of the mismatch- Minor
corresponding tautomeric orMnr
The DNA cleaving function used in our laboratories is EDTAC+GC
groove
still allow the formation of isomorphous
ing pyrimidine base couldgroove
Fe(II), which cleaves the DNA backbone by oxidation ofT-A-T
the
both
to
single
4,
DNA-EDTA-Fe(II)
base
Compared
triplets.
The
deoxyribose with a.2'short-lived diffusible hydroxyl radical (5, 9).T-A-T
CH3 probes 7 and 8 generate weaker cleavage patterns at 0?C
mismatch
~~~~~~~T-A-T
fact that hydroxyl radicalis a relatively nonspecific cleaving species
is
patterns
more apparent for-Rthe cleavage
H
and the difference becomesCH3
Fe
H
~~*T-A.T
useful when studying recognition because the cleavage specificityT-A
is R-Nproduced at 25?C (Fig. 5B). Probes 7 and
R- N8 cleave
/N~the target DNA
N
H>N+
A-T
H,
N,
H,
that
This
result
indicates
H
4.
DNA-EDTAFe(II)
less efficiently than
due to the binding moiety alone, not some combination of cleavage
,
N
N
0
*
~~T-A
N
nucleoin a DNA-EDTAFe(II) Hprobe, 15
a0 singleHbase mismatch
specificity superimposed on binding specificity. The most sequenceA-T
H
N
N
HNthe cleavage efficiency by at least a factor of
*
~~~T-A tides in length, can lower
N
to the natural
specific molecules characterized so far, with regard
0
A-T
doubleNhelical
10. Clearly,
H
</the sequence-specific recognition of large
</~~~~~
product analog approach, is bis(EDTA-distamycin)fumaramide,
*
~~T-A
N
Nojl/Hbase
is sensitive toN single
DNA byNN~
DNA-EDTAFe(II) probes
A-T
which binds in the minor groove and cleaves at sites containing 9 bp
H
R
R correct homoan indication of the importance of the
mismatches,
T-A
of contiguous A,T DNA (10). A synthetic peptide containing 52
pyrimidine probe sequence for the formation of+a triple-stranded
A-T
T
t
TATbase
triplet
complex with the target DNA.
C GCbase triplet
basepairs.
basetriplets
(Top) Watson-Crick
(Bottom)
Isomorphous
DNA. The
of plasmid
double strand
cleavage
Site-specific
Fig. 6. (A) Double strand cleavage of plasmid DNA analyzedon aFig. 1.
TAT and
C'GC. The additional
bound
pyrimidine
1 toChemical
percent
agarose
gel. (Lanesof
3) Plasmid
pDMAG10of ability
at aby Hoogbreaks
double strand
strand is
of DNA-EDTA-Fe(II)
9 to cause
The authors are at nondenaturing
the Arnold and0.9
Mabel
Beckman
Laboratories
Synthesis,
the iscomplementary
linearized
with StyI Engineering,
andlabeledat the
downstream
endof
of the
restrictionsitesteen
hydrogen bonds in the major
Division of Chemistry
and Chemical
California
Institute
Technology,
DNA
insert groove
in large to
presented in purine
homopurine-homopyrimidine
with [a-32P]ATP.(Lanes4 to 6) Sameplasmidwith the otherend-labeled
strand in the Watson-Crick duplex.
Pasadena, CA 91125.
I restriction
with [a-32P]TTP. Cleavageconditions:'P-labeled DNA plasmid,100mM Fig. 6A. The plasmid pDMAG1O was digested with Sty
Figure 1.3. (Left) Simplified model of the triple helix complex between a Hoogsteen bound
DNA-EDTA•Fe probe at single site within 4.06 kb of plasmid DNA. (Right) Isomorphous base
NaCI,1 mM spermine,25 mM tris-acetate,
pH 7.0, 100 pM (bp) sonicated, endonuclease to produce a linear DNA fragment 4.06 kb in size,
30 OCTOBER I987
RESEARCH
1.0 kb
locatedARTICLES
deproteinizedcalf thymus DNA, 5 pM DNA-EDTAFe(II) 9, 25 pM which contains the homopurine site d[A5(AG)5]
64.5
and 2 mM
were run
for 16
hoursat 00C. pyrimidine
Fe(II),
Cleavage
site. This affords
heterogenous overrestriction
upstream from the
triplets
of DTT.
TAT
andreactions
C+GC.
The
additional
strand
is bound
by Hoogsteen
hydrogen
(Lanes1 and4) Controlscontainingno DNA-EDTAFe(II)9. (Lanes2 and hangs and each end could be labeled separately with either
5) DNA size markersobtainedby digestionof Sty I linearizedpDMAG10
to
standard
procedures.
or
according
[a-32P]TTP
with
Eco RI, in
Pvu the
I (both ends
I, Sal major
labeled),and
with [a]2P]ATPpurine
bonds
groove
to Xmn
theI labeled
complementary
strand in the Watson-CrickTheduplex.
[a-32P]TTP:4058 (undigestedDNA), 3338, 2994, 2368, 1690, 1460, 32p end-labeled DNA was allowed to incubate with DNA-EDTAK
1064, and 720 bp. (Lanes3 and 6) DNA-EDTAFe(II) probe9 at 5 pJM Fe(II) 9 (5 pM) for 10 minutes at 0?C as previously described, and
Adapted
from3.04-kb
reference
added.
Arrows indicate
(lane 3) 16.
and 1.02-kb (lane 6) cleavage the cleavagereactionwas initiatedby additionof DTT~(2 mM) and
fragments.(B) (Left) The course resolutioncleavagepatternfrom (A).
at
for 16 hours. Separationof the cleavageproductsby
(Center)Simplifiedmodel of triplehelix complexbetweenthe Hoogsteen- run 0?G
followed
This9content
on Wed,gel12electrophoresis
Jun 2013 01:09:47
AM by autoradiographyreveals
boundDNA-EDTA'Fe(II)
at singledownloaded
site within4.06from
kb of 140.254.87.103
plasmidDNA. agarose
oneConditions
majorcleavagesite producingtwo DNA fragments,3.04
onlyand
(Fig. 4B).
All use subject to JSTOR Terms
RESEARCH ARTICLES
30 OCTOBER I987
649
This seminal study had important implication for the ability of TFOs to induce local
conformational changes
in B-DNA
and more
importantly
regulate gene information
This content downloaded
from 140.254.87.103
on Wed, 12
Jun 2013 01:09:47 AM
All use subject to JSTOR Terms and Conditions
process artificially when equipped with functional moieties. It paved the way that led to
today’s understanding and development of the chemotherapeutic and antigene strategy
based on TFMs. Similar work was also presented by Hélène and co-workers.17 Since
then, TFOs made of native oligonucleotides or their analogs have been extensively
explored to direct DNA triplex assembly.
7
1.3.1.2 Structural Features
Triplex formation via Hoogsteen or reverse Hoogsteen base pairing (Figure 1.4 and
Figure 1.5) is based on the fact that the base pairs in the duplex still have extra hydrogen
bond acceptors and donors available in the major groove for specific recognition by
native nucloebases or their analogs.
300
Copyright # 2001 John Wiley & Sons, Ltd.
Figure 1.4. Hydrogen bonding pattern of base triplets formed by natural nucleobases through
either Hoogsteen (upper left) or reverse Hoogsteen (lower). Base triplets are described as T•AxT,
C•GxC+, C•GxG, and T•AxA with (•) representing Watson-Crick and (x) representing Hoogsteen
or reverse Hoogsteen. TFOs’ strand orientations are indicated by + (parallel orientation with
respect to the homopurine strand ) or – (anti-parallel orientation). An intermolecular DNA triple
helix is schematically represented (upper right) with the third strand TFO in black. Adapted from
reference 1.
8
M. Faria and C. Giovannangeli
J Gene Med 2001; 3: 299–310.
Figure 1. Continues on next page
peptide nucleic acids, PNAs), recognise the oligopurine
strand in the duplex DNA.
Among the three groups mentioned above, TFMs have
been the most extensively used in studies of artificial
modulation of DNA information processing, while small
Bonding rules
Hydrogen bonding interactions keep the two strands
of the DNA double helix together by formation of
Figure 1. Triplex formation. (A) Base triplets are presented: TeArT, CeGrC+, CeGrG, TeArA with (e) representing Watson–
Crick bonding and (r) Hoogsteen and reverse Hoogsteen bonding. Hoogsteen (upper) and reverse-Hoogsteen (lower) hydroFigure 1.5. Classification
of binding configurations of TFOs according to their compositions.
gen bonding patterns are described. The position of the C’1 carbon atom of deoxyribose is indicated by circles containing
either a cross (parallel orientation with respect to the oligopurine strand, e.g. (+) on the purine base of the double helix and
(x) on the
TFO base). An intermolecular triple helix is schematically represented (upper right): the third strand (TFO in black) establishes
hydrogen bonds with the oligopurine strand of the duplex (Pu in grey) in the major groove of the duplex. (B) The binding
modes of the triplex-forming molecules (TFM) are schematically represented. Triplex-forming oligonucleotides (TFO) (left). The
relative orientations of the different TFOs are depicted by arrows (5kp3k) and the compositions of the third strand are indicated [(T,C), (A,G) or (T,G)-motif triplexes]. For (T,G)-containing TFOs the orientation depends on the base sequence [97].
Triplex-forming peptide nucleic acid (PNA) (right). Binding of a bis-PNA to the oligopurine-containing strand and D-loop formation. The preferred orientation is indicated (N terminuspC terminus) for the duplex-forming portion (Watson–Crick bonding, WC) and for the triplex-forming portion which interacts by formation of Hoogsteen hydrogen bonds (Hoog)
The relative orientations
shown
by arrows
to 3’) and
the compositions
of the
third
(+)are
on the
TFO base),
or a line (5’
(anti-parallel
orientation,
e.g. (+) on the purine
base
of thestrand
double helix and
are indicated as (T, C), (T, G) or (A, G). Adapted from reference 1.
Copyright # 2001 John Wiley & Sons, Ltd.
In Hoogsteen approach,
the third strand is bound parallel to the homopurine strandJ Gene Med 2001; 3: 299–310.
whereas anti-parallel in reverse Hoogsteen. However in both Hoogsteen and reverse
Hoogsteen cases, triplex formation must also follow the binding rules originate from
chemical structure of nucleotides and maximum hydrogen bonds formed for energy
reasons. The target sequence must contain homopurine tract because only purine bases
can establish extra hydrogen bonds with incoming third base in the major groove. In
addition, pyrimidine-rich TFOs composed of T and C nucleotides bind in a parallel
orientation via Hoogsteen pattern, forming T•AxT and C•GxC+ base triplets. Such
triplexes are strongly pH dependent as hemi-protonation of C is required. Purine-rich
TFOs composed of A and G bind in an anti-parallel orientation via reversed-Hoogsteen
9
pattern, forming T•AxA and C•GxG base triplets. However, TFOs contain T and G can
bind in either parallel or anti-parallel manners depending on the sequence18 (Figure 1.5).
1.3.1.3. TFOs Modifications
The ability of TFOs made of native nucleotides to be used in biological applications is
largely limited by their chemical and physical properties: (1) target sequence is almost
exclusively purine tract; (2) the requirement of protonation for cytosine especially
adjacent cytosines. In general, this can only be realized in a relatively lower pH
environment than physiological condition; (3) purine-rich TFOs tend to self-assemble
into G-quadruplex or other more complicated structures at physiological ionic strength;
(4) the charge repulsion from sugar-phosphate backbone needs to be neutralized and the
stability of triplex is highly dependent on the presence of relatively higher concentration
of divalent cations such as Mg2+ than that is available in vivo. In order to overcome some
of the limitations listed above, a great number of scientific efforts have been devoted to
modifying the chemical structures of TFOs during the last two decades, including base
modification,
sugar-phosphate
backbone
modification,
covalent
attachment
of
intercalating regents at the 5’ and/or the 3’ end of TFOs, etc. Much progress has been
made toward improving their binding affinity and specificity as well as resistance to
nucleases in a cellular environment.
1.3.1.3.1. Base Modifications
The use of cytosine derivative19,20 for recognition of GC base pair such as 5methylcytosine reported first by Dervan and van Boom21,22 can partially alleviate the pH
10
restriction of TFOs, possibly due to the enhancement of base stacking from methyl group
or exclusion of water molecules from major
groove.
Nonnatural
deoxynucleosides P1
Design of
a Nonnatural
Deoxyribonucleoside
J.
the N 1 and N
separated by co
3a and 3b wer
tallographic a n
protonation requirement for C+GC base triplet formation.23
the methylene
by 8.9% and th
These were assi
were confirmed
3b. Ammonoly
d
A
which in turn
Treatment
J.
Am.
Chem.
Soc.,
Vol.
114,
No.
4,
1992
1471 of 5
Design of a Nonnatural Deoxyribonucleoside
C+GC
which was oxid
I
saturated
the N 1 and N 2 fl anomers, 3a and 3b, which could be with
readily
U
P1.
Nucleoside
separated by column chromatography. The structures of isomers
protection met
3a and 3b were assigned by ’H N M R (NOE) and X-ray
crystreated with di
tallographic a n a l y ~ i s .Irradiation
~
at the C-H1’ proton enhanced
nucleoside Sa.
the methylene proton peaks of ethyl 5-carboxylate of onepropylchloroph
isomer
13.3%.
by 8.9% and the 5-methyl protons of the other isomer byphosphoramidi
These were assigned to 3a and 3b, respectively. These assignments
DMT-protecte
were confirmed by X-ray crystallographic analysis of crystalline
methods.
3b. Ammonolysis of 3a in methanol afforded carboxamide
4a, of
d
A
Synthesis
d
A
which in turn was reduced (H2, 10% Pd/C) to amine
5a.P2. Ol
P1 and
Treatment
of
5a
with
phenyl
isothiocyanate
provided
thiol
6a,12
using
standard
C+GC
Pl*GC
which was oxidized to its corresponding sulfonate. Displacement
istry.14 P1, P2
I
with saturated aqueous ammonium hydroxideI3affords theefficiencies
amine, equ
+
Oligonucleotide
P1.
Nucleoside
P1
was
selectively
acylated
on
N5
by
the
transient
Figure 1.6. Base triplets of C -G-C and P1-G-C. Adapted from reference 23.
deprotected
wi
was
protection methodI4 to afford 7a, and the 5‘-hydroxy group
the novel bases
treated with dimethoxytrityl chloride to give the DMT-protected
and base depro
nucleoside Sa. Reaction of Sa with 2-cyanoethyl N,N-diisoch, 1996, Vol. 24, No. 10
T-T-P1-T-3’ an
propylchlorophosphoramidite afforded 9a, the 5’-DMT-protected
scale for ’H N M
phosphoramidite of Pl.14 Nucleoside P2 and the corresponding
andsame
mass spec
DMT-protected phosphoramidite 9b were prepared by the
chloride, 5 mM MgCl2, 0.1 mM
ribonucleotides
methods.
group of P1 or
e, pH 5.5, was incubated for 5 min
Synthesis of OligodeoxyribonucleotidesContaining Nucleosides
d
A
and the novel
U S1 nuclease. To this mixture was
d
A
P1 and P2. Oligodeoxyribonucleotides10-20 were synthesized
Base Compo
Cl, pH 8.0, and 1 U calf intestinal
using standard solid-phase fi-cyanoethyl phosphoramiditetaining
chem-P1 and
Pl*GC
PP-GC
eaction incubated for an additional
coupled
withP2 afte
istry.14 P1, P2, and the abasic phosph~ramidites’~.’~
P1 and
Figure 1. Base
triplets C +equal
GC, P1.GC
and of
P2-GC.
pyrimidine
re. An aliquot of this mixture was
efficiencies
to those
A, G,For
C,the
and
T phosphoramidites.
analyzed
by en
groove
of the
WatsonCrick
duplex
motif, the third
strand is in the major
10-20,
which
contain
T* (thymine-EDTA),
were
Oligonucleotides
mm column of ODS-Hypersil, 20
oligodeoxyribo
parallel to the purine W-C strand and antiparallel to the pyrimidine
deprotected with 0.1 N Na0H.I’ To examine the stability
of by H
H 5.5) and resulted in the elution of
analyzed
W-C strand.’-3
the novel bases through several machine-assisted coupling
notcycles
chemically
20.2 min and T with a time of 21.2
oligodeoxyribonucleotides,
5’and base
two short
oligodeoxyribo
el-building studies,
wedeprotection,
chose to synthesize
the nucleosides
P1 and
h).
and 5’-T-T-P2-T-3’,
wereP2
synthesized
snake venom
P2 (FigureT-T-P1-T-3’
1). The pyrazole
analogues P1 and
possess a on a 10-pmol
mass spectral
studies.
From the IHphosphatase
NMR
scalehydrogen-bonding
for ’H N M R andpattern
to
donor-acceptor
on one edge
that would
es
Analysis by H
and mass spectral
analysis
of the
purified
tetramer
mimic an N3-protonated
cytosine
and thus
could
form two
hy- oligodeoxygonucleotide 5
drogen bonds
to G in the purine
W - Cfound
strandthat
withthe
no requirement
were performed in 10 mM PIPES,
ribonucleotides,
it was
N-isobutyroyl protecting
sides.
Similarly
for protonation
5ox
amine
group(Figure
of P1 or1).P2 was completely removed to afford a free
7.0, 10.5 mM HEPES, pH
7.5,
Figure 1.7. Hydrogen bonding pattern of m C-G-C
base
triplet.
Adapted
from
reference
24.
T*,
T,
C, and
Proton
N
M
R
,
mass
spectral
analysis,
and
enzyme
digestion
and
the
novel
pyrazole
base.9
or 10.5 mM HEPES, pH 8.5,
d
A
forded
T and P
P1 and P2 nucleosides
areOligodeoxyribonucleotides
stable during the
studies showedBase
that Composition
Analysis of
Conum chloride and 50 mM sodium
and P2 nucleo
oligonucleotide
automated
base d e of
p r the
o t ~ nonnatural
t i o n . The
~
taining
P1 andsynthesis
P2. Theand
integrity
nucleosides
ions in the low micromolar range
PP-GC
nucleotide
P1 forautomated
G C base pairs
is documented
by
sequence specificity
of after
P1
and
P2
synthesis
and
deprotection
steps
was syn
nm) and temperatureFigure
values
1. were
Base triplets C + GC, P1.GC and P2-GC. For the pyrimidine
P1-GC
characterizing
the relative
stabilitiesdegradation.
of PIsGC, P1-AT,
P1.TA
analyzed
by
enzymatic
The
base
composition
of and P
motif, the third strand is in the major groove of the WatsonCrick duplex
DS UV/Visible spectrophotometer
Cleaving.
and P l C G oligodeoxyribonucleotides
triplets by the affinity cleaving
method.
When
incontaining nucleosides P1 and P2 was The
parallel toofthe
all four Watson
found heterocycle
to
corporated analyzed
in a pyrimidine
oligonucleotide,
ure control. The temperature
thepurine W-C strand and antiparallel to the pyrimidine
by
HPLC
to
ensure
thatP1thewas
pyrazole
was
W-C strand.’-3
motif were exa
recognize G C base pairs as selectively and strongly as C but over
ed in 0.5 C steps (from 0 to 95 C)
not
chemically
altered
during
automated
synthesis.
Purified
compared with
an extended
pH range. The utility of P1 is demonstrated in the
m was reached, temperature
Figure
1. (a)
General scheme
for a bidentateP1
hydrogen
donor for the
P2base
were treated
with
el-buildingand
studies, we
chose
to synthesize
the nucleosides
and bondoligodeoxyribonucleotides
same
positi~
site-specific
binding
of a 15 base pair sitecontaining
containingP1
fiveorGC
recognition
of
G-C
base
pairs
by
the
parallel-stranded
motif.
(b)
Putative
both
d. Tm values were determined
snake
venom
phosphodiesterase
and
calf
intestine
alkaline
P2 (Figure
1). The5oxpyrazole analogues P1 and P2 possess
a
11
DNA-cleaving
conpairs
(pH
7.8)
and
a
single
16
base
pair
site
containing
six
5ox
Major
minor
forms of the m C to give the corresponding nucleoside monomers.
m C-G-C base triplet.
and by graphical analysis
of the hydrogen-bonding
donor-acceptor
pattern(c)on
one and
edge
thattautomeric
would
relative stabilit
tiguous G Cphosphatase
base pairs (pH 7.4) in plasmid DNA.
base analog.
(Figure 1.6) designed by Dervan and co-workers were also able to circumvent the
U
Analysis by HPLC of the enzymatic digestion products of olimimic an N3-protonated cytosine and thus could form two hyResults andgonucleotide
Discussion 5’-T*(T)4(PlT)S-3’revealed T*, P1 and T nucleodrogen bonds to G in the purine W - C strand with no requirement
(12) Rousseau
Synthesissides.
of Nucleosides
P1oligonucleotide
and P2. A scheme
for the five-step
Similarly,
5’-T*(T),C(T)4(Pl)6T-3’
afforded
for protonation (Figure 1).
1968, 90, 2661.
ION
syntheses
of
nucleosides
P1
and
P2
is
shown
in
Figure
2.
Ethyl
T*,
T,
C,
and
P1
nucleosides.
Oligonucleotide
5’-TP2TT-3’
Proton N M R , mass spectral analysis, and enzyme digestion
(13)afYamzaki
3-methyl-4-nitropyrazole-5-carboxylate
(1)Io
was condensed
with that3032.
Nucleoside
synthesis
forded T and P2 nucleosides.
These
analyses suggest
the P1
P1
and
P2
nucleosides
are
stable
during
the
studies
showed
that
ard synthetic route from relatively
(14) Oligonuc
(2) to give
and 5ox
P2 nucleosides can be used in automated
oligodeoxyribooligonucleotide automated synthesis and base d e p r o t ~ t i o n .1The
~-chloro-2-d~xy-3,5-di-O-p-toluoyl-a-~-ribofuranose
1984; and referen
The
the phosphoramidite
derivative
ofnucleotide
m C was
appeared to us thatsequence
an effective
synthesis without complications.
P1 for G Cofbase
pairs is documented
by
specificity
of synthesis
(15 ) 5-0-(4,4
generally
withP1-AT,
high P1.TA
yields
(Scheme
1). Thesis,
The
rming stable base triplets
with target theaccomplished
(9) Koh,
J. S.P1-GC
Ph.D.
California
Institute
of Technology,
1990. by N,N-diisopropylp
and P2.GC
Triplet
Stabilities
Characterized
Affdty
characterizing
relative stabilities
of PIsGC,
nucleotides as an
(10) Lewis, A.
heterocyclic base (6-aminothymine) was prepared
byF.;a Townsend,
simple L. B. In Nucleic Acid Chemistry; Townsend,
el-stranded recognition motif should
e plots.
’
McLaughlin and co-workers also reported 2’-deoxy-5-methylcytidine (m5oxC) as an
analog of an N3-protonated cytosine derivative (Figure 1.7).24 When the m5oxC-G-C base
triplets were present in sequence positions that alternate with TAT base triplets, DNA
triplexes were pH independent in the range of 6.4-8.5.
More interestingly, a number of other modified nucleobases and nonnatural analogs have
also been synthesized with respect to expanding the target DNA sequence repertoire by
triplex formation. The ability of TFOs to bind to pyrimidine tracts or hetero purinepyrimidine sequences was addressed (Figure 1.8 and 1.9). One particular strategy
reported by Barry Gold25 was very promising. The use of different modified bases to
specifically recognize T-A vs A-T and C-G vs G-C Watson-Crick base pairing eliminates
the strand polarity issue since the third strand reads in one direction purine information in
the major groove on either strand (Figure 1.8).
12
rimidine motif with third strand parallel to purine strand and (b) purine motif with third strand antiparallel to the purine strand.
Figure 1.8. Hoogsteen hydrogen bonding of antiTA, antiAT, antiCG and antiGC C-glycosides to
ed change in strand polarity and movement of a pyrimidine
one between complementary strands
in duplex
DNA at a
their
Watson-Crick
pair step (darkened bonds represent the attachment to the
backbone).
base pair partners. Adapted from reference 25.
Figure 3. Hoogsteen H-bonding of antiTA, antiAT, antiCG, and antiGC
C-glycosides to their duplex Watson-Crick partners.
J. AM. CHEM. SOC.
9
VOL. 125, NO. 8, 2003 2085
J . Am. Chem. SOC.,Vol. 114, No. 21, 1992 1971
D2 X=H
D3 X=NHCOPh
Figure 1.9. Design
?.CG
Figure 2. Design rationale for recognition of Watson-Crick CG base
pairs by nonnatural bases D2 and D, within a pyrimidine-purine-pyriof rationale
recognition of Watson-Crick CG base
midine
triple-helixfor
motif.
pair by nonnatural
bases D2 and D3 within a pyrimidine-purine-pyrimidine triplex motif. Adapted from reference 26.
fi
tural base triplets T-AT and C'GC. The third-strand
e major groove by Hoogsteen hydrogen bonds to the
on-Crick duplex. Bottom: Novel heterocycles must
cognition of TA and CG Watson-Crick base pairs
e position of the phosphate-deoxyribose backbone in
ine-pyrimidine triple-helix motif.
ng patterns of the pyrimidinepurinepyrimidine
idinapurine motifs, wherein the third-strand base
bonds only to the Watsonxrick purine b a ~ e . ' * ~ , ~
he base 4-(3-benzamidophenyl)imidazole (D3)
ding affinity and sequence specificity for duplex
however from those anticipated by the original
Rather than preferential CG base pair binding,
oth pyrimidinapurine base pairs (TA and CG)
urinepyrimidine base pairs (AT and GC) within
riple-helix motif. Utilizing this expanded rec-
"
1
13
/
RO
R=Tol
R=H
2
3
d
Hd
4
R=R'=H
\P/O-CN
I
7
1.3.1.3.2. Intercalating Reagent
The attachment of an intercalating agent at the 5’ and/or the 3’ end of TFOs can strongly
stabilize triplex formation without sequence-specificity being compromised.27 In the
work presented by Hélène and co-workers, an acridine derivative was covalently linked
to the 5’ end of a homopyrimidine oligonucleotide and specific binding to a homopurinehomopyrimidine DNA duplex was demonstrated. McLaughlin and co-workers also
studied the triplex stabilizing properties of oligodeoxyribonucleotides functionalized at
the 5’ and/or 3’ end with a naphthalene diimide-based (NDI) intercalator (Figure 1.10).28
Thermal denaturation data indicated a remarkable gain in stability for triplex formed
using oligodeoxyribonucleotide-NDI conjugates.
Nucleic Acids Research, 2000, Vol. 28, No. 10 2131
Figure 1. Structure
of the1.10.
3′,5′-NDI
bisconjugate with NDI
intercalators
to both the 3bisconjugate
′- and 5′-termini ofwith
an oligonucleotide.
Figure
Representative
structure
ofconjugated
the 3’,5’-NDI
NDI intercalators
attached to both the 3’ and 5’ end of an oligonucleotide. Adapted from reference 28.
drastically reduce the coupling yield by reacting with the
phosphoramidite. On the basis of HPLC analyses of the products,
this final coupling yield with the NDI (1) was estimated to be
~50%. This procedure produced sufficient material to conduct
analyses for characterization of the conjugate and to determine
Tm values for the corresponding triple helices. Owing to the
relative lability of the diimide conjugates, we employed a fastdeprotecting dC building block for all DNA syntheses
described.
Incorporation of the NDI into the 3′-terminus of a support-bound
oligonucleotide should have been relatively simple. Modified
supports such as the amino support provide both an Fmoc-protected
confirm that essentially all of the amino groups had reacted and
then we capped the support only after addition of the first
nucleoside residue.
14
TFOs with intercalators modification at the middle of the sequence (Figure 1.11) was also
contemplated by McLaughlin and co-workers.29 In this context, the perylene- and
naphthalene diimide-based intercalators were employed as the linker of two
homopyrimidne segments, thus providing conjugates capable of targeting single-stranded
Naphthalene and Perylene Hairpin Triplexes
J. Am. Chem. Soc., Vol. 122, No. 25, 2000 5909
nucleic acids with the formation of hairpin triplexes. The intercalators were designed to
bridge the terminal base triplet and participate in base-stacking with all three residues.
Figure 1. (a) Hairpin triplex with five dT residues tethering the two pyrimidine binding sequences. (b) Complex illustrated in (a) with a nonnucleosideFigure
linker tethering
two binding
sequences. (c) A DNA-naphthalene
diimide conjugate.
(d)A
A DNA-perylene
DNA-perylene diimide
conjugate.
1.11. to
(Upper)
A DNA-naphthalene
diimide conjugate;
(Lower)
diimide
Scheme 1conjugate. Adapted from reference 29.
remaining tBDMS-protecting group) could be obtained with a
limited HF/pyridine reaction. The mono-tBDMS-protected
product (6) could then be converted to the DMT-protected
derivative 7. The second tBDMS-protecting group was then be
removed, and the resulting product (8) converted to the
phosphoramidite derivative (9).
The naphthalene linker was prepared in a similar but more
straightforward manner. After incorporation of the two tBDMS
linkers,
the tBDMS-protecting groups were both removed, and
1.3.1.4. Triplex-forming Peptide Nucleic Acids
(PNAs)
the product was converted successively to the mono-DMT
derivative and then to the corresponding phosphoramidite.
Oligonucleotides are difficult to prepare in large Sequences
scale (millimole
mole quantities)
of DNA or
or 2!-O-methyl
RNA, orand
appropriate
chimeric sequences were prepared in the conventional manner
on CPG solid-phase
At present
the site ofmajor
the linker, the
introduction of modified nucleobases and conjugation
to othersupports.
ligands
naphthalene-based phosphoramidite was incorporated in much
that same manner as a nucleoside derivative. The perylene
problems. Besides, poor cellular uptake of oligonucleotides
hastoto
a large
extent
limited
derivative continued
exhibit
poorer
solubility
characteristics,
and to avoid clogging the lines of the DNA synthesizer, the
30
was performed
with the
CPGcolleagues
in a small flask.
After
the potential of TFOs in molecular biology coupling
and medicine.
Nielsen
and
this coupling reaction, further elongation in the normal manner
was also not effective. The first coupling after incorporation of
suggest replacing the deoxyribose phosphate backbone
a polyamide
backbone
the perylenewith
diimide
needed to be
performedand
in a 50:50
dichloromethane:acetonitrile solution. Presumably the solubility
of the tethered
wasterms
problematic
acetonitrile. After
the resulting peptide nucleic acid (PNA) is homorphous
to perylene
DNA in
of theinnumber
the coupling reaction, and the removal of the excess linker, the
15 support became yellow (naphthalene) or burgundy (perylene)
in color. After completion of the synthesis, the CPG beads were
treated with concentrated methanolic ammonia (naphthalene)
or concentrated aqueous ammonia (perylene) overnight. After
isolation by PAGE, each complex was analyzed by HPLC with
detection at 260 nm and either 383 nm (naphthalene) or 537
in aqueous solutions (quite likely worse), but after incorporation
nm (perylene). All of the conjugates eluted as single peaks under
into sequences of DNA, the polyanionic nature of the DNA
both conditions of detection.
resulted in a significant improvement in aqueous solubility of
We were concerned about the stability of the diimide linkages
the conjugates.
of backbone bonds and the distance between backbone and nucleobase. The targeting
acridine lig-TFO
at significant
DNA-intercalating
melting
temperature
Tm the
(temperature
poses.was
The similar
concept here
to conventional
approach
in that
third strand
(either protection
of the A
and was expected to increase the affinity for which 50% of double-stranded DNA is de- quence (Fig. 3A), whereas n
double-stranded DNA as demonstrated for natured) of the hybrid between dAjo and was observed on the T strand
PNA or DNA)
would
bind to the
purine(6),
target
strand
hydrogen
was via
and that between
PNA-1
86'CHoogsteen
these results, increased cleav
conjugates
oligonucleotide
analogous
dAjo bonding.
strand of the target sequence
and the nitrobenzamido ligand of the acri- and PNA-2 (Fig. 1) was 73?C.
Binding of PNA-1 to double-stranded coccus nuclease was seen in th
dine was expected to make it possible to
The very study
first the
generation
of homopyrimidine PNAs H-T -Lys-NH2 (Figure 1.12
and
DNA binding by affinity photo- DNA was studiedn with a 32P-end-labeled
PNA-1 (Fig. 3B), whereas t
cleavage (7). Furthermore, the helix-thread- DNA fragment containing the dA1o-dT1O increased cleavage of the A st
30-32 and lococcus nuclease cleaves si
of the acridine
After irradiation
expected to bytarget
sequence
design prepared
Table 1.1)ingwere
andwasevaluated
Nielsen
and(9).colleagues.
The Lysine
place the polyamide moiety in the major subsequent piperidine treatment, preferen- DNA [at least in DNA loops
groove and the nitrobenzamido ligand in tial DNA-nicking was observed in the dA1o erence to double-stranded DN
residue at the
C-terminus
was
the purpose
solubility,
electrostatic
attraction
asthe very strong bind
the highest
helix whenof aqueous
result and
minor groove
of for
the DNA
sequence, with
efficiency at Al
binding to double-stranded DNA. The ly- (numbered from the 5' end) and at T2 of to dAjO (Fig. 2) surprisingly
sine ligand was included to give some elec- the complementary strand (Fig. 3, A and B, the binding of PNA-1 to a do
well as preventing
PNAs from self-aggregation. Tm results showed that the designed
trostatic attraction and to increase aqueous and Fig. 4). These nicking results are con- DNA target results in binding
sistent with the binding of PNA-1 along the by Watson-Crick hydrogen bo
solubility.
tract that is
orientwith oligonu- dA1o-dT1o
The hybrids
interactionwith
of PNA-1
strand (the A
complementary
preferentially
PNAs formed
complementary
purine strands.
The
complex
stability
was
cleotides was assayed in two ways. Binding ed with the acridine ligand at the 5' end of displacement of the nonco
to a complementary dA1o sequence was the dA1O. However, the minor cleavage at strand (the T strand). This
strongly susceptible
to gel
theretardation
presence(Fig.
of 2A),
mismatch
bases
A13 and
indicates thatsequence
demonstrated by
T10 indicating
should render the T strand se
binding alsoselectivity.
which also illustrates lack of binding to the occurs with the opposite orientation.
The by UV
were
conductnoncomplementary
Footprinting
experiments
The binding
stoichiometrydT1o
was sequence.
determined
titration
to be 2:1
PNA:
DNA, which
results also show that PNA-1 is able to ed to support the sequence-preferential
1 1 1 1 1 1
32P-oligo
displace the oligonucleotide T strand in a binding indicated by the photo-nicking exoligo2
-- - + + +
was the same
with
TFO
approach except
oligothymidine
was
employed
form
of double-stranded
dA-dT
twofold
excess
oligonutracts are to
sub- triplex.
periments. Oligo
poor
PNA-1 - + + - + +
1 2 3 4 5 6
strates for both deoxyribonuclease I and the
cleotides, indicating that the PNA-DNA
A
hybrid is more stable than normal double- chemical footprinting reagent methidium
stranded B-DNA. The PNA-DNA affinityis propyl EDTA-Fe(II); therefore staphylococso high that the duplex is reformed after cus nuclease and a photo-nicking diazoPNA-DNA
denaturation in 80% formamide (Fig. 2B). linked acridine derivative (10) were used.
complex
With conditions under which a normal Photofootprinting of the PNA-DNA comdA1o-dT1o hybrid melted at 23?C (8), the plex with the diazo-linked acridine showed
SsDNA
dsDNA
0
0
HO
B
Rl-HN
B
N
00
0
0
p
\0H
B
0
B
HNN
0\00 4i
p
0
-0
L~~~
~~~~~~0
0
HN-
B
B
ssDNA
8N
DNA
PNA
OH
Figure 1.12. Generic chemical structure of PNAs (B = thyminyl) studied.NH-R2
The structure of DNA
0
is shown for comparison. Adapted from reference 30.
Fig. 2. Binding of PNA-1 tO
single-strandedDNA; dsDNA, d
N
H2NOC
PNA-1 R1=
;
R2=H
I
NH3+
(CH2,
H6NOC
PNA-2: R1=
-
H
16
o
N
;T R2= H
(CH.2)4'
NH3.
Fig. 1. Chemical structures of PNA-1 and PNA-2 (B thyminyl) (17). The structureof DNA is shown
for comparison.
1498
DNA). 5 '-32P-labeled oligonucl
GATCCA1,0G) (16) (lanes l to 6)
in the absence (lanes 1 and 4)
PNA-l (lanes 2 and 5,,25 pmol; la
pmol), and in the absence (lan
presence (lanes 4 to 6) of olig
(5'-GATCCT10G).5 '-32P-labele
tide 2 (lanes 7 to 9) was incubated
(lane 7) or in the presence of PNA
pmol; lane 9, 75 pmol). The sam
lyzedby PAGEandautoradiogra
conditions (A) or denaturing cond
presence of PNA-l and oligo 2
(+); absence is indicated by (-)
SCIE
Communications fo the Editor
(2)"
Table 1.1. Thermal Stabilities of PNA-DNA triplexes. Adapted from reference 31.
Table I
'NH-R'
2
N
o
n
0
R2 = H, n
, R2 = Acr,
eous NaOH
v) BocNH-
PNA
DNA
Tma
H-T6-Lys-NH2 ( 2 4
(dA),
31
H-Tg-Lys-NH, (2b)
(dA),
52
H-TIO-LYS-NH~
( 2 ~ ) (dA)lo
72d (a)
H-TIo-Lys-NH2 ( 2 ~ ) (dA)lo
72b
H-TIO-LYS-NH~
(2C)
(dA)lo
73c
H-Tlo-Lys-NH2 ( 2 ~ ) (dA)s(dG)(dA)r
59d (b)
H-Tlo-Lys-NH2(2c)
(dA),(dG)(dA),(dG)(dA),
46d (c)
Acr-Tlo-Lys-NH2
(dA),,
86
"The melting temperatures of the hybrids were determined on a
Gilford Response apparatus. The following extinction coefficients were
used: A, 15.4, T? 8.5, and G, 11.7 mL/gmol.cm for both normal deoxyoligonucleotides and PNA. The solutions were 10 mM in phos10 mM in MgClZ,
140 mM
in NaC1,
pH 7.4, unless otherphate,characterization
Further
of the binding
between
PNA H-Tand
8-Lys-NH2 to a complementary
wise stated. 0.3 OD260/mLof 2c (for 2b 0.24 OD,,/mL and for 2a
single-strand
poly(dA)was
chainhybridized
by flow linear
dichroism
(LD)
and circular
dichroism
(CD)
0.18 OD,,/mL)
with
1 equiv
of the
other strand
(using
the extinction coefficients above for both PNA and DNA) by heating
confirmed
discrete
PNA-DNA
assembly
with 2:1 PNA:
binding
to 90 "Cthefor
5 min,
coolingtriplex
to room
temperature,
andDNA
storing
forratio
30
min followed by storage at 5 OC for at least 30 min. The melting
and that it was a right-handed helix.32 The base conformation of the poly (dA)-PNA2
curves were recorded in steps of 0.5 OC/min. The T, values were determined
from
the tomaximum
of the firstpoly
derivative
of the plot of A,,
triplex
was very
similar
that of the conventional
(A•T-T) triplex.
versus temperature.
mM MgCI,, 0 mM NaCI.
mM MgC11, 500
However,
quite
surprisingly,
PNAs
of
sequence
H-T
-Lys-NH
was
not able
to form the
mM NaC1. dThe melting curves for a, b, nand c are
in Figure
1.
2 shown
expected DNA-DNA-PNA triplex with dA10-dT10 duplex tract, but rather by Watson-
1
Crick hydrogen bonding to the complementary A strand and displacing the
1.6 (Figure 1.13).30,33
noncomplementary T strand
Normalized
Absorbance
i
17
Temperature
B
1
- + + - + + --+
C
+ PNA-1
A
5 50 500 5 50 500A
+ + +
300 nm KMnO4 c
staph A/G
1 2 3 4 5 6 7 8 9 1011
1
2
3
4
5
6
S
PNA-1
7
B
300-nm Radiation
photocleavage
5 -GRTCCRARRAARARRGGATC
3' -CTAGGTTTTTTTTTTCCTAG
D'azoacridine
photofootprlnt
5' -GRTCCAAAAAARARAGGATC
3' -CTRGGTTTTTTTTTTCCTAG
+t
l
sU|6
4nmzl
VtStaphylococcus
nuclease
enhancement
5'-GRTCC ARRARROAGGRTC
3'-CTRGGTTTTTTTTTTCCTAG
. 1.
~
~~~~~~~~~
KMnO4 cleavage
T
ATT
AT
-A T
T
T
T
T
-AT
A
T
AT
T
AT
T
AT T
-AT T
5 -GRTCCRAAARRARRRGGATC
3' -CTRGGTTTTTTTTTTCCTAG
ItP S1-nuclease
cleavage
Figure 1.13. Schematic representation of strand displacement PNA binding mode upon targeting
....^.3
A10
~
~
.1
of the probFig. 4. (A) Schematicrepresentation
signifies
length
of the arrows
ing results.The
double stranded DNA. Adapter
from
reference
30.
::i.:'
w
j
*
|
AT3
T,00
4.0~~~~~~~~~~4
cleavage intensity. The quantitationwas performedby densitometricscanningof the autoradiogramsin Fig. 3 and by subtractingthe corresponding background controls. For 300-nm
photocleavage,lanes2 in (A) and 2 in (B) were
used with lanes 1 in (A) and 1 in (B) as background.Lanes5 in (A) and6 in (B) wereusedfor
Lane5 in (B), withlane
DNA photofootprinting.
4 in (B) as background,was used for KMnO4
enhancement.Lane9 in (B), with lane8 in (B) as
nuclease
wasusedforstaphylococcus
background,
enhancement,and lane 5 in (C), with lane 2 in
(C) as background,was used for nuclease SI
enhancement.The bracketindicatesthe region
protected from photocleavageby the diazolinked-acridine.(B) Cartoonof the PNA-1-dsDNA strand-displacement
complex.
In order to address the ability of PNA to invade the local duplex, displace the
*
.
**,4
*
S
oligopyrimidine strand and meanwhile form an internal extremely stable triplex structure
(triplex invasion binding mode),34 Egholm et al.35 designed a type of dimeric PNAs (bis-
emical, and enzymatic probing of the dsDNA-PNA-1 complex. Either the A
d (B and C) was probed. Complexes between PNA-1 and a 32 P-end-labeled
g a dlA10-dT10target sequence (9) were probed by affinity photocleavage (A
, and 120 pmol of PNA-1, respeatively); photofootprinting (A, lanes 5 and
1, respectively); potassium permanganateprobing (B, lanes 4 to 6, 0, 40, and
ectively); or probing by staphylococcus nuclease (B, lanes 8 to 10, 0.,40, or
ectively) or by nuclease S1 (C, lanes I to 3, no reagent; lanes 4 to 6, 120 pmol
0.005 U/mi SI concentration, shown at IOOOXin figure; lanes 2 and 5, 0.05
U/mi). For lane 4 in (A), the photocleavage was performnedwith the free
. In (B), lane 7 served as no-treatment control. The A+ G sequence reactions
(B) lane 11, and (C) lane 7.
positively charged lysine increase nonspecific DNA affinity and ensure a high local
concentration of the PNA close to the
DNA. Thus, strand displacement can be
initiated through inherent DNA breathing
(13) and proceed in a zipperlike fashion.
This mechanism is supported by the observation that strand-displacement binding of
PNA-1 to the target sequence, as probed by
potassium permanganate hyperreactivity, is
a slow process in which maximum reactivity
is only observed after more than 202 min.
We believe that the high stability of the
PNA-DNA hybrids is due to the lack of
electrostatic repulsion between the two
strands combined with the constrained flexibility of the polyamide backbone of the
PNA. Although this backbone has a limited
number of energetically favorableconformations because of the presence of planar
amido groups, it has high flexibility at the
aminoethyl linkers.
These results should apply to mixed sequences with the other three DNA bases,
PNAs) where two monomeric PNA segments were covalently connected together via a
flexible linker (Figure 1.14). One segment was designed for Watson-Crick recognition of
DNA and the other designed for Hoogsteen recognition of PNA-DNA duplex.
and-specificnuclease proposed strand-displacement binding
othermost
thisstrandshouldbe mode, and we
are not aware of anythe
Consequently,
onby potassiumper- binding mechanism that would account for
ngof PNA-1, all thyquencecould be oximanganate
(Fig. 3B),
mentary
strand of the
pecificallyattackedby
ed a symmetricaldisntensitiescenteredat
no increasedcleavage
ryA strandwas seen.
e consistentwith the
them. The acridine ligand is suspendible,
but the Tm values show that it increases
PNA-DNA stability (12). Furthermore, the
observed preferred polarity of the binding
may be due to the presence of the acridine.
Although stand displacement must be
thermodynamically favored because of the
higher stability of the PNA-DNA hybrid
compared to normal double-stranded DNA,
it is surprising that it takes place so readily.
We believe that the acridine ligand and the
stable DNA-PNA triplex would be formed when the Watson-
Crick base-pairing PNA segment is bound to the DNA strand in the anti-parallel
orientation whereas the Hoogsteen strand is in the parallel orientation.33
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1499
18
mentioned above, TFMs have
used in studies of artificial
tion processing, while small
Hydrogen bonding interactions keep the two strands
of the DNA double helix together by formation of
Figure 1.14. Triplex-forming peptide nucleic acid (PNA). Binding of a bis-PNA to the
oligopurine-containing strand and D-loop formation of the oligopyrimidine strand. The preferred
orientation is indicated by arrows (N: N terminus; C: C terminus; WC: Watson-Crick; Hoog:
Hoogsteen). Adapted from reference 1.
Base triplets are presented: TeArT, CeGrC+, CeGrG, TeArA with (e) representing Watson–
een and reverse Hoogsteen bonding. Hoogsteen (upper) and reverse-Hoogsteen (lower) hydrobinding
dataatom
indicated
that bis-PNAs
were able
formcontaining
triplex with oligonucleotides
ribed. The position of The
the C’1
carbon
of deoxyribose
is indicated
by to
circles
ion with respect to the oligopurine strand, e.g. (+) on the purine base of the double helix and
in a sequence-specific
manner,
thermal
stability
ne (anti-parallel orientation,
e.g. (+) on the purine
baseofof somewhat
the doublehigher
helix and
(x) on
the than monomeric
riple helix is schematically represented (upper right): the third strand (TFO in black) establishes
purine strand of the duplex
grey)
in the
major transition
groove of showed
the duplex.
The
binding Furthermore, bisPNAs(Pu
andinthe
thermal
melting
very(B)
little
hysteresis.
olecules (TFM) are schematically represented. Triplex-forming oligonucleotides (TFO) (left). The
erent TFOs are depictedPNAs
by arrows
(5kp3k) and
the compositions
of the third
strandofare
indialso exhibited
superior
binding properties
in terms
targeting
double-stranded
otif triplexes]. For (T,G)-containing TFOs the orientation depends on the base sequence [97].
acid (PNA) (right). Binding of a bis-PNA to the oligopurine-containing strand and D-loop forDNA through
triplex
mode.
on is indicated (N terminuspC
terminus)
forinvasion
the duplex-forming
portion (Watson–Crick bondming portion which interacts by formation of Hoogsteen hydrogen bonds (Hoog)
A reasonable explanation for the enhanced triplex stability and rate of strand invasion
Ltd.
J Gene Med 2001; 3: 299–310.
was proposed by Griffith and co-workers36: the entropy cost accompanying the assembly
was essentially reduced and the binding reaction was converted into a bimolecular
process by simply linking two PNA segments together. In their research work with
similar bis-PNA designs , where a neutral polyethylene glycol (PEG) or a positively
19
charged lysine/aminohexyl were both investigated as the linker (Figure 1.15). The
binding affinities of these bis-PNAs for single-strand (ss) DNA and binding rates for
(ds) DNA
were significantly increased. Quite impressively,
the
J. Am. double-strand
Chem. SOC. 1995,117,
831-832
83 1
stoichiometry of a 1:1 PNA-DNA complex was established by direct observation on
B
Nucleic Acids as Triplexing
oichiometry
electrospray mass spectrometry (ESI), indicating the robust stability of PNA-DNA
M. Risen, Michael J. Greig,
triplex.
Sprankle, Richard H. Griffey,
. Freier
maceuticals, 2280 Faraday Avenue
Carlsbad, California 92008
Received August 26, 1994
m
Above is a generic PNA structure with amino acid end-groups
to enhance solubility and stability. B is the nucleobase unit (N1alkylated cytosine, N9-alkylated adenine, etc.).
nucleic acids (PNAs) form stable
H-Gly-TTC-TCT-CTC-T-Lys-Aha-Lys
d (ss) oligonucleotides and invade
HzN-Lys-TTC-TCT-CTC-T-Lys- AhaL
The resulting triplexes consist
DNA strand. A number of oligoatic functions are inhibited by P N N
3
H2N-LyS-TTC-TCT-CTC-T-Gly-H’
xes, including restriction enzyme
Ac-Lys-Aha-Lys
se transcription, and tran~lation.~-~ 4
H2N-Lys-TTC-TCT-CTC-T-Lys-AhaL
As attractive agents for oligonucle‘written carboxy to amino for sequence comparison
ics.
RNtukw
vasion at physiological-level ionic
A
65mer duplex containing target site: 5 ’ - - - -AAGAGAGAGA- - - - -3’
ssed in order to further the use of
3’- - - m C r T C T C T -- _ _ - -5’
tools and antigene agent^.^ One
B
65“
duplex containing target site: ~ ’ - - - - - A G A G A G A G A A - - - - - ~ ’
strands together to reduce entropy
(reverse orientation)
3’-----TCTCTCTCTT------5’
imolecular process. Egholm et al.
o N-terminus to C-terminus linked
C
single stranded target
5’-TAAGAGAGAGATGTTA-3’
ormation.8 We have prepared bisD
single stranded target
a neutral (poly(ethy1ene glycol)(reverse orientation)
ed (lysine/aminohexyl) linker and
Figure 1. PNA and DNA sequences. Aha, 6-aminohexanoic acid;
s to determine the relative affinities
3PEG,
H2N(CHzCH20)3CH2COOH.i4
1.15. The
studied
PNA and DNA sequences. Aha, 6-aminohexanoic acid; 3PEG,
NAs for ds and ss DNA. Figure
Additionchiometry of the complexH2between
N(CH2CH2O)3Table
CH2COOH.
Adapted
from of
reference
1. EC50
for Binding
PNA to 36.
Double- and Single-Strand
observation via electrospray mass
DNA Targets“
__
Strand Invasion
n Figure 1 were synthesized by
target B
PNA
target A
alyzed by HPLC and electrospray
-PNA 1 containing the positively
1
50 nM
40 nM
tially better strand invasion
of
the
2
22
p
M
p M on-rate and slow offKinetics of strand invasion
study demonstrated
a relatively 40
rapid
3
28 p M
65 p M
gle PNA 3 or the PEG-linked bis-
4
6 PM
6 PM
Berg, R. H.; Buchardt, 0. Science
1991, was consistent with the mechanism for PNA invasion of ds DNA proposed by
rate, which
Single-Strand Binding
vskii, B. P.; Frank-Kamanetskii, M. D.;
37,38
target“locks”
D
PNA
R. H.; Nielsen, P. E. Proc. Nielsen
Natl. Acad.and colleagues.
The secondtarget
PNAC strand essentially
the association
1
30020
pM
275 pM
Buchardt, 0.;
Berg, R. H. J. Am. Chem.
2
225 pM
250 pM
3
20 nM
12 nM
Berg, R. H.; Buchardt, 0. Nucleic Acids
4
100 nM
200 nM
; Berg, R. H.; Buchardt, 0. Anti-Cancer
a PNA binding was measured using a gel mobility assay.15 We define
ECso as the PNA concentration at which 50% of the target is bound.
; Bisi, J. E.; Thomson, S. A.; Cadilla, R.;
Radiolabeled target (20 pM) was incubated for 3 days (ds target) or 1
man, C. F.; Bonham, M. A.; Au, K. G.;
; Boyd, A. L.; Noble, S. A.; Babiss, L. E.
day (ss target) with increasing concentrations of PNA at 37 “C in 100
mM Na+, 10 mM phosphate, and 0.1 mM EDTA, pH 7.0 (charge
duplex target (1, 2) at low ionic strength.
Further study showed a tendency for
poly(T) PNA to form 2:1 PNA-DNA
process in a triplex upon binding, which indeed was the rate-limiting step.
complexes (3). Several groups have deInterestingly, the crystal structure of a nine-base bis-PNA-DNA triplex (Figure 1.16)
signed and synthesized bis- or hairpin
revealed
a P-form helix,
which differed from
previously reported
A-form like based
formation
byon
PNAs
to promote
triplex
the tethering
original fiber diffraction
of poly(dA)-[poly(dT)]PNA
triplex. strands
This important
twostudies
polypyrimidine
These
bygained
flexible
(4,
5).
finding
insight intolinkers
the manner in
which
PNAs
complexedPNAs
with DNA did
to form
indeed
have
affinity
singleincreased
triplexes.
The observed
hydrogen
bonding geometries
and distancesfor
of T•A-T
and C•G-C
stranded
and
higher
rate
ofthestrand
triplets
provided directDNA
confirmation
of the a
proposed
recognition
pattern:
purine base
invasion for double-strandedDNA, relafrom each triplet forms a Watson-Crick base pair with the pyrimidine base on one PNA
39
deter
hairp
The
stand
acids
and r
2
strand and a Hoogsteen base pair with the pyrimidine on the other PNA strand. Moreover,
A
this unique structure expanded the repertoire of known stable helical forms that nucleic
Baseclasses of stable DNA
acids can adopt and it also suggest that there may be
X>additional
>3
>
CF
\ X2
CD
triplexs yet to be discovered.
\
CG
y
Xi,CE?.,
NB
CA
CT
II
a
B
1
*
9
C-T TTCTC
CTTC
chrC
is
PNA
strand
3
Figure 1.16. Diagram of the bis-PNA-DNA triplex. Adapted from reference 39.
Watson-Crick+H N-
,,,,
DNA
I,,,,
3-GAGAAGAAG-5'
Hoogteen -OOC_________
TTCTTC
JCTC
PNAstrand
T
21
24
Gly . 12
Ser
Ser
is ply
16
Fig. 1. PNA monomer and PNA2-DNA1complex
structures. (A) Structure of a PNA monomer.
Backbone torsion angles are indicated by Greek
letters according to convention 1). Carbon po-
Table
DNA
by an
an eq
0.1 M
40C (
reserv
Cryst
c =
-1 70
a cry
25%
DENZ
obtain
ture o
plates
two io
cated
by an
phase
the P
done
rame
phase
resolu
into th
gram
and b
The novel binding modes presented by PNA and its synthetic availability have made this
molecule a highly attractive ligand toward sequence specific DNA recognition.
Nonetheless, only homopyrimidine PNAs have been explored extensively in triplexforming field so far. This is largely due to the underlying Hoogsteen-mediated targeting
concept. Thus, much more improvement is yet to be made, especially concerning the
ability to target pyrimidine or mixed sequences, before full potential of PNAs can be
accessed and evaluated. And this goal can be potentially achieved by expanding the
library of the attached nucleobases and their analogs through chemical modification.
1.3.2. Novel Janus-Wedge Approach
A Janus-Wedge (J-W) recognition concept was first proposed by Lehn and colleagues40
in their work with heterocycles. It is based on the design of wedge-shaped heterocycles
presenting two hydrogen-bonding arrays that are complementary to the Watson-Crick
faces of a pair of canonical or mismatched nucleobases and thereby forming a basewedge-base triad motif (Figure 1.17).
In this initial work, the incoming third heterocycle was able to recognize a cytosine-uracil
mismatched base-pair by insertion in a wedge-like fashion utilizing the maximum
number of Watson-Crick interactions (Figure 1.18). However, the first generation wedges
1a and 1b designed were lipophilic and all binding properties were characterized in
CHCl3.
22
lizing the maximum number of bind by inserting between the two nucleobases (Scheme 1). The
In order to avoid these interferences one may target not the
ns, as shown by 1H NMR spectro- resulting
formation
fiveand
or GC
six hydrogen
bonds
canonical
base-pairsof AT
but, instead,
pairs should
of
provide
high
binding
affinity
and
specificity.
However,
since
mismatched bases which are present in important structural
presenting
recognition
surfaces
comfeaturesheterocycles
such as mutated
sites of DNA
and unpaired
regions of
s of nucleic acids may be recognized JANUS
to those
of the
two nucleobases
in apurine-purine,
natural pair, are
RNA forming
bulges
or loops.
Of the possible
nd in the major or minor groove of the plementary
overall yield throug
cyanoacetate and 3,
lective hydrogenatio
condensation with gu
The binding prope
assessed by analysing
on titration7of a CDC
purine-pyrimidine and pyrimidine-pyrimidine mismatches, the
e between the base-pairs. In contrast
cytosine-uracil pair is a particularly attractive target. Crystal
aromatic n-stacking interactions of
structures of double helical nucleic acids show that, unlike e.g.
inor groove binders like oligonucleothe guanine-adenine5 and the guanine-uracil6 'wobble ' pairs
etropsin derivatives,3 present a more
which form two hydrogen bonds, the cytosine-uracil pair
nce specificity through the formation
exhibits only one direct and one water-bridged hydrogen bond.6
onds. In this approach, base-pair
The JANUS molecules l a and lb, differing only in the position
subtle differences in number and
of the organic solubilizing groups, are CU-wedges designed to
bonding sites in the grooves of the
insert between cytosine and uracil so as to yield a total of six
uence, a complete set of DNA binders
'R
hydrogen bonds upon formation
of the ternary CWU complex.
natural base-pair (AT, TA, GC and
Additional stabilization may be provided by enhanced aromatic
eloped.
n-stacking interactions due to the increased area of the planar
approach towards base-pair recognitriad.
t. It is based on the design of wedgeThe lipophilic wedges l a and l b were synthesized as
enting two hydrogen bonding arrays
R
outlined in Scheme 2.$ Compound
lva (white solid, mp
the Watson-Crick faces of a pair of
290-293 OC, R f 0.36 in CH2C12-10%MeOH-1%Et3N on silica)
ched nucleobases. By analogy with
NUS heterocycles4 presenting two
was obtained in one step from commercially available starting
', they are referred to Figure
as JANUS
materials
the concept:
reaction
ofJANUS-type
2-amino-6-chloropyrimidin-4-ol
1.17. Lehn’s
Janus-Wedge
wedges wedges
with two
hydrogen
bonding faces (A:
Scheme
1 Theby
WEDGE
concept.Janus
with
two hydrogen
n god depicted with two faces on bonding
with 3,5-di-tert-butylaniline
in
a
refluxing
mixture
of
acetic
and
(A: acceptor,
D:
donor)
arebetween
designedbase-pairs
to bind byforming
insertiona triplet with
acceptor,
D: donor)
arefaces
designed
to bind
by yield
insertion
ad. Instead of targeting
hydrogen
hydrochloric
(28%
recrystallization
between
base-pairsacid
forming
a triplet
withafter
the maximum
number of from
WatsonEtOH).
base-pairs as realized inthe
most
of the Crick
l b (white
solid, mp
Rf0.41 in
maximum
number
ofDerivative
Watson-Crick
interactions.
In192-195
contrast,
conventional
DNA binding
interactions.
In contrast,
common
DNA
binding"C,
molecules
target
40% (or
the JANUS wedges are designed to hydrogen
CH2C12-10%MeOH-1
%Et3N
on
silica)
was
prepared
in black
bonding sites of the intact base-pair, as illustrated by
molecules
target
hydrogen
bonding
the(or
intact
base-pair,
as illustrated
by
black (or small
overall
through
aofKnoevenagel
condensation
of AT
ethyl
the two nucleobases (Scheme
1). The
for sites
the major
minor)
groove
side
of the
basesmall
open) yield
arrows
ve or six hydrogen bonds should pair.cyanoacetate and 3,4-didecylbenzaldehyde, followed by seopen) arrows
the major
(or minor) groove
of the
ATsubsequent
and GC base-pair.
nity and specificity. However,
since for lective
of theside
alkene
and
cyclo- Adapted from
hydrogenation
esenting recognition surfaces
comcondensation with guanidine.
reference
40.
The binding properties of the CU-wedges l a and l b were
two nucleobases in a natural pair, are
assessed by analysing the lH NMR spectral changes occurring
on titration7of a CDC13 solution of l a or l b (1 0 mM) with a 1 : 1
a
'R
R
k
v
cwu
BU'
pt. JANUS-type wedges with two hydrogen
: donor) are designed to bind by insertion
iplet with the maximum number of Watson, common DNA binding molecules target
intact base-pair, as illustrated by black (or
jor (or minor) groove side of the AT base-
Chern. Commun., 1996
2443
Figure 1.18. Lehn’s Janus-Wedge molecule capable of forming a triad with thymine and cytosine
derivatives in CHCl3. Adapted from reference 40.
23
k
McLaughlin and his co-workers41 followed up the investigation on this novel targeting
concept and reported the first example of Janus-Wedge type DNA triplex formation. In
the design, eight J-W bases (Wedge residue W) were attached to a PNA backbone,
capable of base pairing with the Watson-Crick faces of cytosine-thymine mismatch sites
in the DNA sequence 11dC8-11/11dT811, leading to the formation of eight (C-W1-T)
base triplets (Figure 1.19). A Lysine (K) residue was added at the C-terminus to afford
aqueous solubility as well as complementary charge-charge interaction. Experiments
suggest that the resulting PNA W8K bound to the DNA target in the major groove,
parallel to the dC8 strand and antiparallel to the dT8 strand. It was noteworthy that
thermal denaturation of the J-W triplex exhibited two-phase transitions, corresponding to
triplex-duplex and duplex-random coil equilibrium respectively. Thermodynamic
analysis confirmed that the J-W triplex was stabilized by a free energy of 15.2 kcal/mol,
which was substantially greater than the conventional TFOs approach of similar sequence
length.
They soon sought out to investigate the ability of J-W type PNAs to undergo strand
invasion where the wedge residues were involved in hydrogen bonding with the WatsonCrick faces of canonical base pairs (A-T, G-C).42 In the study, a wedge residue W1 was
used to target A-T (or T-A by simple rotation of heterocycle), and W2 for G-C (or C-G)
base pairs (Figure 1.20).
24
istry, Merkert Chemistry Center, Boston College, 2609 Beacon Street,
Chestnut Hill, Massachusetts 02467
ceived August 22, 2003; E-mail: [email protected]
lity of a Janus-Wedge
from two DNA target
base pairing with the
ences.
y a third strand binding
e of purine residues.1,2
groove of duplex DNA
ne sequences using a
G-C base triplets) or
Published on Web 09/11/2008
base triplets) probe
Figure 1. (a) A Janus-Wedge base triplet: the third-strand residue W binds
ional approach is that
Figure 1.19.
(a)W-C
A Janus-Wedge
base
triplet:
the third
residue
W binds
the Watsonto the
faces of both
target
residues,
(b)strand
the 11dC
11-11T
target
811 to
ets.
challenge
to withsequence.
dgeThe
DNA
Triplex
A-W1-T and G-W2-C Base Triplets 8
faces of both target residues; (b) the 11dC8-11/11dT811 DNA target sequence. Adapted
pyrimidines within Crick
a
Han Chen, Meena,
and
Larry
W. McLaughlin*
To41.
probe for complex formation, the W8K oligomer was added
g approaches includefrom reference
of Chemistry, Merkert ChemistrytoCenter,
College, 2609 Beacon
Street, as well as to the duplex
each ofBoston
the single-stranded
target strands
gen bond,4 recognition
Chestnut Hill, Massachusetts 02467
itself, and the mixtures were analyzed by nondenaturing PAGE
to bind a purines in
Received
June
17,
2008;
E-mail: [email protected]
(Figure
2). The two DNA single strands are found in Lanes 5 and
ng approaches7-9 have
6, while in Lanes 1 and 2 one equivalent of the W8K strand has
of a Janus-Wedge (J-W) triple been added to each. The T -target strand is more effectively shifted
8
ecognition
motif
base pairs. Each
basefirst
triplet is
to a lower mobility species (Lane 2) than is the dC8-target strand
rocycles,
hird residueinvolves
(a wedgethe
residue)
drogen
bond with
theof the (Lane 1) suggesting more effective binding to the former. Mixing
he Watson-Crick
faces
the 11T811 and 11dC811 strands resulted in a lower-mobility species
get strands (see Figure
and Helene
formed from a (Lane 4) interpreted to be the duplex of Figure 1b.
about
this are
recognition
Addition of one equivalent of the W8K strand to the duplex target
to the Hoogsteen
of purine
based
upon theface
triplet
1,2
air. With that design the third resulted in complete shift of the duplex band to a lower-mobility
J-W triplex be formed
of duplex DNA. The Dervan/ species that we interpret as being the J-W complex (Lane 3).
?butHere
we answer the
is generally limited to the
The gel shift experiment indicates that complex formation occurs,
1,2
nces (with the formation of but it cannot clarify the nature of binding. It appears that the W K
8
ageneralize
J-W triplex
wetargeting,
pre- a
duplex
strand binds more effectively to the T8 strand than the dC8 strand
d whereby
all four possible base
with
11 Watson-Crick
ements
Dervan/Helen (compare Lanes 1 and 2 of Figure 2), and in the triplex it could, in
). The over
two the
pyrimidine
principle, bind only to the T8 strand. We performed a chemical
ng derivatives
thattoemploy
edge
base (W)
both only
4
of each base pair as a unit, or probing experiment with bromide and monoperoxysulfite known12
such as5 that illustratedFigure 1.20. Janus-Wedge
Figure 1. Janus-Wedge base triplets: (a) the third-strand residue W1
base
triplets: (a) the third-strandand
residue W1 binds to the W-C
faces of
strand, but improvements in to differentiate
between
binds to the W-C
faces ofsingle-stranded
the target A and T or (b)double-stranded
W2 binds similarly dC
g
a
purine-pyrimidine
only moderate successes.target A and T;
to (b)
G and
C.
W2 binds
to G and C similarly. Adapted from reference 42.
ry.
In
this
initial
study,
x is based upon a recognition
to
with W-C
to generate the aminotriazenedione ring involved attack of a secondary
rk compete
with heterocycles;
it involves
amine on a CBz protecting group.13 Fmoc deprotection unmasked a
amino-pseudocytidine
nd
to hydrogen bond with the
primary amine; we reasoned this amine could attack the remaining
wo
target strands
Figure 1).
ackbone.
The (see
peptide
CBz protecting group (forming a 10-membered ring, see Supporting
Wt1 isto neutral
target A-T
by simple
and(orlimits
25
Information). Mass spectral analysis confirmed this possibility. Replacpairs as we described earlier,10
quences can bind DNA
ing that CBz protecting group with trimethyl-phenylsulfonyl (Scheme
We have used a neutral PNA
hould
be
advantageous
1) eliminated the putative side reaction, and PNA sequences containing
ve incorporated one lysine (K)
ueous solubility and to provide
inker
wasThe
attached
teractions.
choice ofto
a PNA
at
certain PNA sequences are
minopyrimidin-4-one.
m
D-loop structures,
a form of
otected
as the CBZ
W1 and W2 could be prepared.
We prepared a mixed 8-mer PNA sequence with a terminal lysine
residue: (W1)3W2(W1)4K, purified it (HPLC), and confirmed its
mass. In previous work10 we had also prepared the (W1)8K PNA
A series of mixed 8-mer PNAs with a terminal lysine residue of sequence
(W1)m(W2)l(W1)nK were prepared and tested against a number of different DNA
sequences (Table 1.2). Tm values supported relatively high binding affinities and
selectivities. However the PNA 8-mer was unable to form a detectable J-W triplex when
the target sequence contained A-T and G-C base pairs (i.e. no mismatch sites C-T), likely
due to the reason that eight-residue PNA was not long enough to effectively invade the
target duplex and form stable triplex. A longer sequence (W1)14K targeting dA14/dT14
duplex indicated the formation of a stable complex, although it was not clear whether the
observed complex was actually J-W triplex.
Table 1.2. Thermal stabilities of J-W triplexes. Adapted from reference 42.
Table 1. Thermal Stabilities of J-W Triplexes
J-W Triplexes
X-Y
wedge strand
TM
-X X X X X X X X-
T-C
T-A
T-C
G-C
W1W1W1W1W1W1W1W1K
W1W1W1W1W1W1W1W1K
W1W1W1W1W2W1W1W1K
W1W1W1W1W2W1W1W1K
45.8, 69.2 ((0.5) °C
44.9, 69.1 ((0.5)
36.4, 70.1 ((0.3)
((0.3)
C O 46.9,
M M U69.8
NIC
ATIONS
-C C C C C C C C-C A C C C C C C-C A C C C A C C-C A C C C A C A-A A A A C A A A-
Table 2. Thermal Stabilities of J-W Triplexes
TM
45.8,
44.9,
36.4,
46.9,
69.2
69.1
70.1
69.8
((0.5) °C
((0.5)
((0.3)
((0.3)
tember 24, 2009 | http://pubs.acs.org
, 2008 | doi: 10.1021/ja804607v
W1W1K
W1W1K
W1W1K
W1W1K
Table 2. Thermal Stabilities
nces in which T-C was used for the
ed for the target of W2 (Table 1).14
the (W1)8K probe and the T-C target
latter transition corresponds to the
m and the former characterizes the
ntroduction of one W2 residue into
ent a mismatch condition for the all
M characterizing the triplex-duplex
ased by roughly 9 to 36.4 °C (Table
W2 site was changed to the canonical
W2, the TM value characterizing the
also prepared two DNA sequences in which T-C was used for the
target for W1 and G-C was used for the target of W2 (Table 1).14
The observed TM values for the (W1)8K probe and the T-C target
was 45.8 -Xand
69.2
to the
XXX
X X X°C.
X- The latter transition corresponds
TM
duplex-random
coil
equilibrium
and
the
former
characterizes
the
-C C C C C C C C46.9, 69.8 ((0.3) °C
triplex-duplex
Introduction44.3,
of one
2 residue into
-C A C C equilibrium.
C C C C69.1 W
((0.4)
-C A
CCC
A C C34.2, 67.6
((0.7)for the all
the probe
strand
should
represent a mismatch
condition
-C A and
CCC
C A19.9, 67.2
T-C target,
in Afact
the TM characterizing
the ((0.7)
triplex-duplex
-A A A A C A A A- 78.7
equilibrium in this case decreased by roughly 9 to 36.4 °C (Table
1). When the T-C target at the W2 site was changed to the canonical
triplex
as pair,
-15.3thekcal/mol
(50WmM
NaCl,
10 mM
MgCl2). While
G-C
base
target for
TM value
characterizing
the
2, the
this value reflects
effective rose
binding
interactions
through
the J-W
triplex-duplex
equilibrium
by over
10 to 46.9
°C (Table
1).
format,
it
will
be
moderated
when
strand
invasion
is
required.
It is noteworthy that placing a26
single W2 residue into a sequence
of the
PNA sequence
W2 residue
the
thatBinding
targets T-C
residues
results in containing
a significantthe
decrease
in TMto
value
target
DNA
containing
a
G-C
base
pair
(Table
1)
was
also
and that thermal stability is recovered when the G-C target is place
confirmed
nondenaturing
PAGEAn
analysis,
which resulted
in a
to
interact by
with
the W2 residue.
examination
of potential
K
of
12
nM
(Figure
3).
D
hydrogen bonding interactions for the T-W2-C base triplet suggests
the formation of an essentially isomorphic base triplet (Figure 2),
but the existence of such a base triplet does not contradict the data.
The measured TM values simply indicate a loss in complex thermal
triplex as -15.3 kcal/mol (
this value reflects effective
format, it will be moderated
Binding of the PNA sequ
target DNA containing a
confirmed by nondenaturing
KD of 12 nM (Figure 3).
Figure 3. Gel shift assay for (
Initial studies with a long
T14 duplex have indicated t
The J-W recognition mode proposed by Lehn and the J-W triplex strategy later developed
by McLaughlin and co-workers were fundamentally novel directed towards the sequencespecific recognition of DNA and the accompying triplex assembly. In contrast to most of
the traditional DNA recognition motifs, the J-W are designed to bind by inserting
between two nucleobases rather than targeting hydrogen-bonding sides of the intact base
pairs presented in either minor or major grooves. It elegantly solved the long-held
challenge presented by conventional TFMs with homopurines as the sole targets. The
promising results from their study may also shed a light on the future design of
recognition elements capable of targeting all four base pairs with enhanced affinity and
selectivity. However, the studied DNA target sequences were all preformed duplexes. It
was highly possible that the stability of formed J-W triplex was dependent on WatsonCrick base-pair brackets at both ends. Therefore, it will be of significant importance to
understand in greater detail the stabilization energy contributed solely from Waston-Crick
hydrogen bonding at the recognition interfaces as well as to explore J-W recognition
motif on a more synthetically convenient scaffold.
27
Chapter 2
Experimental: Design, Synthesis and
Characterization
28
2.1. DNA Triplex-forming Peptide Design
Nucleic acid triplex structures formed from native oligonucleotides are known to occur
via purine Hoogsteen base-pairing of a third strand in the major groove of a WatsonCrick base-paired duplex.16,43,44 This has been extensively developed by Dervan45 and
others46 as a targeting concept using both native and artificial nucleobase recognition
elements and backbones. McLaughlin42 and Tor47 have both reported elegant “Januswedge”40 recognition of Watson-Crick interfaces using synthetic nucleobases on PNA
and sugar-phosphate backbones, respectively. While these prior methods have largely
sought to develop general strategies for sequence targeting of preformed oligonucleotide
structures, there are fewer synthetic approaches to generate structure in single-stranded
oligos or effect triplex formation without prior oligonucleotide duplex assembly; such
structure-inducing recognition may be useful in design of synthetic regulators of
transcription48,49 or translation.50-53 We have explored this notion using a Janus-wedge
approach to address two identical interfaces: two oligothymidine (dT10) DNA tracts were
assembled on a peptide template via bifacial melamine recognition to form peptide-DNA
triplex structures. Compounds closely related to melamine and its canonical hydrogenbonding partner, cyanuric acid,54-56 have been used to site-substitute for native
nucleobases in PNA-DNA duplex recognition. Melamine itself is a well-known
molecular recognition module in a number of contexts,57-66 and Baranger and
Zimmerman67 have recently reported melamine targeting of thymine-thymine or uraciluracil (T-T, U-U) mismatch sites in d(CTG) and r(CUG) repeats, assisted by acridine
29
intercalation.68-72 Indeed, the hydrogen-bonding pattern of melamine precisely
complements the Watson-Crick face of thymine/uracil (Figure 2.1).
Figure 2.1. Bifacial melamine-thymine recognition. Synthetic peptides 1-4 present melamine and
methylated melamine on derivatized lysine side chains. Peptide 2 is N-terminated with (5,6)carboxyfluorescein (Cbf) and β-alanine (β-Ala). Putative peptide-DNA complex structures are
shown: triplex 5 and hairpin 6.
We recently characterized the recognition of cyanuric acid and melamine derivatives at
aqueous interfaces73-76 as well as bulk solution77 and found that binding was strongly
dependent on the number of heterocycles per scaffold: a trivalent system yielded robust
binding while monovalent recognition was undetectable. Based on this prior work, we
hypothesized that multivalent presentation of melamine heterocycles on a peptide
backbone would bind two strands of dT10 into a peptide-DNA heterotrimeric bundle.
Indeed, though dT10 has no detectable homo-oligomerization behavior, synthetic
30
melamine-displaying peptide 1 induced assembly of a peptide-DNA triplex structure in
heterotrimeric stem and heterodimeric hairpin systems with a peptide-DNA ratio of 1:2
and 1:1, respectively (Figure 2.1).
Prior studies from Eschenmoser and Krishnamurthy54,56 and Ghadiri78 demonstrated the
use of α-peptide backbones, instead of the traditional PNA backbone,42,79,80 to recognize
DNA. This method forms a recognition interface from alternate residues and was more
synthetically convenient for our purposes. We introduced melamine through side-chain
functionalization of Boc-Lysine with chlorodiaminotriazine, to yield a derivative we term
melaminolysine (M*). Boc deprotection with TFA followed by reaction with Fmoc-OSu
yielded Fmoc-M*, which was used in standard solid-phase peptide synthesis (SPPS) with
a C-terminal glycinamide (Figure 2.1). Alternate residues were glutamic acid to provide
water solubility and to avoid nonspecific electrostatic binding to DNA, yielding peptide
1, (EM*)10G. This sequence was N-terminally capped with carboxyfluorescein (peptide
2) to permit fluorescence-based binding analysis. Control peptides were also synthesized
in which hydrogen-bonding sites were systematically blocked by methylation of the
exocyclic amines on the melamine ring. Stepwise chloride displacement of
trichlorotriazine81 with ammonia and/or dimethylamine and Boc-lysine yielded the
dimethylated and tetramethylated melaminolysine derivatives, which were used in SPPS
to provide peptides 3 and 4, respectively.
31
2.2. Materials and Methods
2.2.1. General
Chemicals for amino acid derivatives synthesis, peptide synthesis, purification and
characterization were purchased from Sigma-Aldrich and used without further
purification unless otherwise specified. Rink Resin LS (100-200 mesh) was purchased
from Advanced ChemTech. DNA Oligomers were obtained from Sigma-Aldrich except
5’-Cy5-dT10 from Integrated DNA Technologies, Inc. Dye-labeled DNA oligomers were
HPLC purified and the unlabeled ones were purified by standard desalting. All DNA
oligomers were quantified at 260 nm on UV-Vis HP 8543 using the extinction
coefficients provided by the manufacturers. 40% acrylamide and bis-acrylamide solution
(29:1) was purchased from Bio-Rad. TBE Buffer (10X solution) for gel electrophoresis
was purchased from American Bioanalytical. Dulbecco’s Phosphate-Buffered Salines
(DPBS 1X, 2.67 mM KCl, 137.93 mM NaCl, 1.47 mM KH2PO4 and 8.06 mM Na2HPO4,
pH 7.4, no calcium, no magnesium) used as the binding buffer for all the binding studies
discussed in this chapter was purchased from Invitrogen.
MALDI-Mass spectra were acquired on Bruker Microflex MALDI-TOF instrument
under RP mode. Electrospray mass spectroscopy was accquired on a Bruker MicroTOF
equipped with an electrospray ionization source under positive mode. Mass spectrometry
instruments were provided by a grant from the Ohio BioProducts Innovation Center.
NMR spectra were acquired on a Bruker Advance DPX 400 instrument.
32
2.2.2. Synthetic schemes for monomer preparation
NH2
Cl
N
NH2
H2N
O
N
N
HN
NH2
NaOH/H2O
N
H
O
N
COOH
N
N
NH2
a) TFA
b) Fmoc-OSu NaHCO3
O
85 °C overnight
N
H
O
1,4-dioxane/H2O
COOH
0°C-R.T. overnight
1a
NH2
N
HN
N
N
NH2
O
O
N
H
COOH
1
Figure 2.2. Synthesis of monomer 1.
NH2
O
N
Cl
NH3 H2O
N
N
Cl
O
NH
NH2
Cl
N
Cl
/ H2O
N
NH2
NaOH
N
N
O
Cl
/ H2O
O
Cl
N
NaOH/H2O
N
85 °C overnight
3b
3a
NH2
NH2
N
HN
N
N
N
HN
N
a) TFA
O
b) Fmoc-OSu NaHCO3
O
O
N
H
COOH
N
R.T. 24h
0 °C-R.T. 1h
N
H
COOH
1,4-dioxane/H2O
O
N
H
0°C-R.T. overnight
3
3c
Figure 2.3. Synthesis of monomer 3.
33
COOH
N
N
N
NH2
O
Cl
N
Cl
NH
N
N
N
O
Cl
/ H2O
O
N
NaOH
Cl
N
H
COOH
N
N
N
NaOH/H2O
85 °C overnight
R.T. 0.5h, 50 °C 1h
4a
N
N
N
HN
N
N
N
HN
O
N
O
b) Fmoc-OSu NaHCO3
N
H
N
N
a) TFA
O
N
COOH
1,4-dioxane/H2O
O
N
H
COOH
0°C-R.T. overnight
4b
4
Figure 2.4. Synthesis of monomer 4.
2.2.3. Synthetic procedures
(S)-2-((tert-butoxycarbonyl)amino)-6-((4,6-diamino-1,3,5-triazin-2yl)amino)hexanoic acid (1a).82
Boc-Lys-OH (7.389 g, 30 mmol) was dissolved in water (80 ml) and was added to a
water suspension (20 ml) of 6-chloro-1,3,5-triazine-2,4-diamine (5.24 g, 36 mmol).
NaOH (2.4 g, 60 mmol) in 60 ml water was slowly added to the mixture and the reaction
was stirred overnight at 85 °C. The reaction mixture was cooled down to R.T. and white
solid was filtered off. The aqueous solution was acidified to pH 5 with HCl (1 M
solution) at 0 °C. The resulting precipitate was filtered and washed with water (2x15 ml)
and dried under high vacuum to give 10.1g (yield 95%) of 1a as a white solid. 1H NMR
34
(400 MHz, d6-DMSO) δ (ppm) 1.21-1.70 (m, 15H), 3.15 (m, 2H), 3.83 (m, 1H), 6.14 (br
s, 2H), 6.30 (br s, 2H), 6.62 (t, J = 5.6 Hz, 1H), 6.91 (d, J = 7.9 Hz, 1H). 13C NMR (100
MHz, d6-DMSO) δ (ppm) 23.05, 28.25, 28.95, 30.79, 53.74, 77.92, 155.57, 165.71,
166.52, 174.78. HRMS (ESI) m/z calculated for [M+H]+: 356.2041, found 356.2038.
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((4,6-diamino-1,3,5-triazin-2yl)amino)hexanoic acid (1). 54
Compound 1a (10 g, 28 mmol) is dissolved in TFA (80 ml) and water (1 ml) was added.
The mixture was stirred at R.T. until complete by ESI and TLC (~1h). Maximum amount
of TFA was removed under reduced pressure. The residue was resuspended in 200 ml
water and the solution was neutralized to pH 7 using NaHCO3. Another 1.5 equv. of
NaHCO3 was added and the resulting solution was stirred at 0 °C for 20 min. Fmoc-OSu
(14 g, 42 mmol) in 200 ml dioxane (also cooled) was added slowly. The resulting mixture
was stirred at 0 °C for 1 h and allowed to warm up to R.T. overnight. Water (150 ml) was
then added and dioxane was removed under reduced pressure. The aqueous layer was
washed with EtOAc (2x100 ml), acidified to pH 1 with HCl (1 M solution) at 0 °C and
extracted with EtOAc (3x120 ml). The combined organic layer was concentrated under
vacuum. The resulting residue was purified by flash chromatography (SiO2) with a
solvent gradient from 5 to 10% MeOH in CH2Cl2 to give compound 1 as a white solid (8
g, yield 60%). 1H NMR (400 MHz, d6-DMSO) δ (ppm) 1.34 (m, 2H), 1.50 (m, 2H),
1.58-1.76 (m, 2H), 3.24 (dt appearing as q, J1 = 6.7 Hz, J2 = 6.4 Hz, 2H), 3.93 (m, 1H),
4.22 (m, 1H), 4.27-4.30 (m, 2H), 7.31 (dd appearing as t, J1 = 7.4 Hz, J2 = 7.4 Hz, 2H),
7.40 (dd appearing as t, J1 = 7.4 Hz, J2 = 7.4 Hz, 2H), 7.61 (d, J = 8 Hz, 1H), 7.70 (s,
35
2H), 7.71 (d, J = 7.6 Hz, 2H), 7.72 (s, 2H), 7.88 (d, J = 7.5 Hz, 2H), 8.09 (t, J = 5.5 Hz,
1H). 13C NMR (100 MHz, d6-DMSO) δ (ppm) 22.98, 28.27, 30.45, 46.71, 53.84, 65.65,
120.14, 125.32, 127.12, 127.68, 140.75, 143.85, 156.23, 173.93. HRMS (ESI) m/z
calculated for [M+H]+: 478.2197, found 478.2192.
2-amino-4,6-dichloro-[1,3,5]-triazine (3a). 82
Cyanuric chloride (4.98 g, 27 mmol) was dissolved in acetone (38 ml) and poured into 40
ml of iced-water to form a very fine suspension. Ammonium hydroxide solution (6.76 g
28 %, 54 mmol) was added dropwise at 0 °C. The mixture was stirred at 0 °C for 30 min
and R.T. for 30 min. White solid was filtered, washed with water (4x30 ml) and dried
under high vacuum giving pure compound 3a (3 g, yield 70%). 1H NMR (400 MHz, d6DMSO) δ (ppm) 8.54 (bs, 2H). 13C NMR (100 MHz, d6-DMSO) δ (ppm) 166.96, 169.20.
HRMS (ESI) m/z calculated for [M+H]+: 164.9729, found 164.9722.
N2,N2-dimethyl-2,4-diamine-6-chloro-[1,3,5]-triazine (3b). 82
Compound 3a (2.47 g, 15 mmol) was dissolved in acetone (33 ml) and poured into 42 ml
iced-water to form a very fine suspension. A water solution of dimethylamine (1.69 g
40%, 15 mmol) was added at 0 °C. NaOH solution (15 ml, 1N) was added dropwise at 0
°C. The reaction mixture was stirred at R.T. for 24 h. The white solid was filtered,
washed with water (4x15 ml) and dried under high vacuum to afford pure (2.2 g, yield
85%) 1H NMR (400 MHz, d6-DMSO) δ (ppm) 3.03 (s, 6H), 7.24 (br s, 2H). 13C NMR
(100 MHz, d6-DMSO) δ (ppm) 35.90, 35.96, 164.96, 166.55, 168.37. HRMS (ESI) m/z
calculated for [M+H]+: 174.0541, found 174.0545.
36
(S)-6-((4-amino-6-(dimethylamino)-1,3,5-triazin-2-yl)amino)-2-((tertbutoxycarbonyl)amino)hexanoic acid (3c).
Compound 3c was synthesized from compound 3b and Boc-Lys-OH following the
procedure used for synthesis of compound 1a. 1H NMR (400 MHz, d6-DMSO) δ (ppm)
1.28-1.37 (m, 11H), 1.45 (br s, 2H), 1.52-1.69 (m, 2H), 2.99 (s, 3H), 3.01 (s, 3H), 3.19
(br s, 2H), 3.83 (m, 1H), 6.27 (br t, 1H), 6.65 (br s 1H), 6.78 (br s, 1H), 7.01 (d, J = 7.8
Hz, 1H).
13
C NMR (100 MHz, d6-DMSO) δ (ppm) 23.09, 28.26, 28.78, 30.54, 35.74,
53.52, 78.05, 155.68, 174.36. HRMS (ESI) m/z calculated for [M+H]+: 384.2354, found
384.2348.
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((4-amino-6-(dimethylamino)1,3,5-triazin-2-yl)amino)hexanoic acid (3).
Compound 3 was synthesized from compound 3c following the procedure used for
synthesis of compound 1. Column chromatography (SiO2) purification with a solvent
gradient from 2 to 5% MeOH in CH2Cl2 provided compound 3 as a white solid (yield
53%). 1H NMR (400 MHz, d6-DMSO) δ (ppm) 1.35 (m, 2H), 1.47 (br s, 2H), 1.57-1.76
(m, 2H), 2.98 (s, 6H), 3.19 (br s, 2H), 3.92 (m, 1H), 4.20-4.29 (m, 3H), 6.01 (br s, 1H),
6.12 (br s 1H); 6.50 (t, J = 5.5 Hz, 1H), 7.32 (dd appearing as t, J1 = 7.4 Hz, J2 = 7.4 Hz,
2H), 7.41 (dd appearing as t, J1 = 7.4 Hz, J2 = 7.4 Hz, 2H), 7.61 (d, J = 8 Hz, 1H), 7.72
(d, J = 7.4 Hz, 2H), 7.89 (d, J = 7.5 Hz, 2H). 13C NMR (100 MHz, d6-DMSO) δ (ppm)
23.09, 28.86, 30.52, 35.52, 46.65, 53.83, 65.58, 120.08, 125.27, 127.05, 127.62, 140.69,
143.79,143.84, 156.15, 165.60, 174.02. HRMS (ESI) m/z calculated for [M+H]+:
506.2510, found 506.2518.
37
N2,N2,N4,N4-tetramethyl-2,4-diamine-6-chloro-1,3,5-triazine (4a).82
Cyanuric chloride (4.98 g, 27 mmol) was dissolved in 35 ml acetone and poured into 50
ml ice-water to form a very fine suspension. A water solution of dimethylamine (6.08 g
40%, 54 mmol) was added at 0 °C. NaOH (54 ml, 1N) was added dropwise at 0 °C. The
mixture was stirred 30 min at R.T. and additional 1 h at 50 °C. The solution was cooled
down in ice-water and precipitate was filtered, washed with water (4x30 ml) and dried
under vacuum to afford white solid (4.88 g, 90%). 1H NMR (400 MHz, d6-DMSO) δ
(ppm) 3.04 (s, 6H), 3.06 (s, 6H). 13C NMR (100 MHz, d6-DMSO) δ (ppm) 35.72, 35.81,
164.24, 168.04. HRMS (ESI) m/z calculated for [M+H]+: 202.0854, found 202.0857.
(S)-6-((4,6-bis(dimethylamino)-1,3,5-triazin-2-yl)amino)-2-((tertbutoxycarbonyl)amino)hexanoic acid (4b).
Compound 4b was synthesized from compound 4a and Boc-Lys-OH following the
procedure used for synthesis of compound 1a. 1H NMR (400 MHz, d6-DMSO) δ (ppm)
1.37-1.64 (m, 15H), 3.00 (s, 12H), 3.19 (dt appearing as q, J1 = 6.3 Hz, J2 = 6.3 Hz, 2H),
3.83 (m, 1H), 6.53 (t, J = 5.7 Hz, 1H), 6.99 (d, J = 7.9 Hz, 1H). 13C NMR (100 MHz, d6DMSO) δ (ppm) 23.05, 28.19, 28.90, 30.65, 35.44, 53.58, 77.84, 155.53, 165.23, 165.48,
174.28. HRMS (ESI) m/z calculated for [M+H]+: 412.2667, found 412.2658.
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((4,6-bis(dimethylamino)1,3,5-triazin-2-yl)amino)hexanoic acid (4).
Compound 4 was synthesized from compound 4b following the procedure used for
synthesis of compound 1. Column chromatography (SiO2) purification with a solvent
gradient from 1 to 3% MeOH in CH2Cl2 provided compound 4 as a white solid (yield
38
50%). 1H NMR (400 MHz, d6-DMSO) δ (ppm) 1.37 (m, 2H), 1.53 (m, 2H), 1.57-1.79
(m, 2H), 3.02 (s, 6H), 3.05 (s, 6H), 3.28 (m, 2H), 3.93 (m, 1H), 4.18-4.31 (m, 3H), 7.32
(dd appearing as t, J1 = 7.4 Hz, J2 = 7.4 Hz, 2H), 7.41 (dd appearing as t, J1 = 7.4 Hz, J2 =
7.4 Hz, 2H), 7.62 (d, J = 8 Hz, 1H), 7.71 (m, 3H), 7.89 (d, J = 7.6 Hz, 2H).
13
C NMR
(100 MHz, d6-DMSO) δ (ppm) 22.92, 28.00, 30.34, 36.07, 46.63, 53.61, 65.57, 120.07,
125.21, 126.99, 127.59, 140.69, 143.77, 156.10, 173.88. HRMS (ESI) m/z calculated for
[M+H]+: 534.2823, found 534.2823.
2.2.4. Solid phase peptide synthesis and characterization
Peptide synthesis was performed on an AAPPTEC Apex 396 Peptide Synthesizer using
Rink Resin LS (100-200 mesh, loading 0.2 mmol/g) employing standard Fmoc chemistry
utilizing DIC/HOBt as the coupling agents and 50% piperidine/NMP for Fmoc
deprotection. Peptides were cleaved from the solid support and tert-Butyl protective
groups removed using a TFA : water : m-cresol = 95:2.5:2.5 mixture (3mL for 0.15 g
resin) for 3.5 h at R.T. to yield peptides with amide at C-termini. Resin was removed by
filtration through cotton and the peptides were precipitated by chilled ether (30 ml),
centrifuged, and washed with chilled ether (3x20 ml). Crude peptides were then dissolved
in solvent A and purified by HPLC on a C18 reversed phase column using a gradient of
10–100% solvent B in 50 min (solvent A = 0.1% TFA in water, solvent B = 0.01% TFA
in 45% acetonitrile, 45% isopropanol, 10% water). The UV detector was set at 230 nm.
The purified peptides were lyophilized to dryness. The identity of peptide was checked
by MALDI-TOF and purity checked by analytical HPLC on a C18 column.
39
A
(b)
B
2E+5
Absorbance (230 nm)
1.5E+5
1E+5
5E+4
0
0
10
20
30
40
50
Time (min)
Figure 2.5. (A) MALDI-TOF of peptide 1: m/z calculated for [M+H]+Avg: 3738.839, found
Figure S1. (b) HPLC trace of of peptide 1 on a C-18 analytical column using a gradient of 10-100%
solvent
B over
monitored
by aaUV-Vis
at 230column
nm. (The using
small peak
at 40 min was
3738.181; (B) HPLC
trace
of50ofmin,
peptide
1 on
C-18 detector
analytical
a gradient
of due
10-to
the aggregation of peptide 1 on column. The fraction has the same mass as peptide 1. It was also observed
on 3350
minmin,
peak reinjection
HPLC.)
100% solvent B over
monitored
by a UV-Vis detector at 230 nm. (The small peak at 40
min was due to the aggregation of peptide 1 on column. The fraction has the same mass as
peptide 1. It was also observed on 33 min peak reinjection HPLC.)
40
A (a)
(b)
B
6E+5
Absorbance (230 nm)
5E+5
4E+5
3E+5
2E+5
1E+5
0
0
10
20
30
40
50
Time (min)
Figure S2. (a) MALDI-TOF of peptide 2: m/z calculated for [M+H]+Avg: 4168.217, found 41
Figure 2.6. (A)
MALDI-TOF
peptide2 on
2: m/z
calculated
forcolumn
[M+H]+using
Avg: 4168.217,
HPLC
trace of ofofpeptide
a C-18
analytical
a gradientfound
of 10-100% solvent
min,
monitored
by
a
UV-Vis
detector
at
230
nm.
4168.641; (B) HPLC trace of of peptide 2 on a C-18 analytical column using a gradient of 10100% solvent B over 50 min, monitored by a UV-Vis detector at 230 nm.
41
(a)
A
(b)
B
7E+5
Absorbance (230 nm)
6E+5
5E+5
4E+5
3E+5
2E+5
1E+5
0
0
10
20
30
40
50
Time (min)
Figure S3. (a) MALDI-TOF of peptide 3: m/z calculated for [M+H]+Avg: 4019.370, found 4019
+
Figure 2.7. (A)HPLC
MALDI-TOF
peptide3 3:
calculated
forcolumn
[M+H]using
trace of ofofpeptide
on m/z
a C-18
analytical
a gradient found
of 10-100% solvent B
Avg: 4019.370,
min, monitored by a UV-Vis detector at 230 nm.
4019.118; (B) HPLC trace of of peptide 3 on a C-18 analytical column using a gradient of 10100% solvent B over 50 min, monitored by a UV-Vis detector at 230 nm.
42
-
(a)
A
(b)
B
5E+5
Absorbance (230 nm)
4E+5
3E+5
2E+5
1E+5
0
0
10
20
30
40
50
Time (min)
Figure S4. (a) MALDI-TOF of 5(6)-Carboxyfluorescein (Cbf) labeled peptide 3: m/z calcula
+
Figure
2.8. (A)
of 5(6)-Carboxyfluorescein
(Cbf)
labeled
3: m/z calculated
[M+H]
: 4448.748, found
4448.875; (b) HPLC
trace
of ofpeptide
Cbf labeled
peptide 3 on a C-18 an
AvgMALDI-TOF
+ using a gradient of 10-100% solvent B over 50 min, monitored by a UV-Vis detector at 23
column
for [M+H]
Avg: 4448.748, found 4448.875; (B) HPLC trace of of Cbf labeled peptide 3 on a C-18
analytical column using a gradient of 10-100% solvent B over 50 min, monitored by a UV-Vis
detector at 230 nm.
43
-S
A
(b)
B
5E+5
Absorbance (230 nm)
4E+5
3E+5
2E+5
1E+5
0
0
10
20
30
40
50
Time (min)
Figure 2.9. (A) MALDI-TOF of peptide 4: m/2z calculated for [M+H]2+Avg: 2149.45,2+found
Figure S5. (a) MALDI-TOF of peptide 4: m/2z calculated for [M+H] Avg: 2149.45, fou
2148.64; (B) HPLC trace of of peptide 4 on a C-18 analytical column using a gradient of 10HPLC
trace of of peptide 4 on a C-18 analytical column using a gradient of 10-100% so
100%
solvent
B over
monitored
by a UV-Vis
min, monitored
by45a min,
UV-Vis
detector
at 230detector
nm. at 230 nm.
44
2.2.5. General sample preparation protocol for binding studies
Samples were prepared in DPBS 1X buffer (2.67 mM KCl, 137.93 mM NaCl, 1.47 mM
KH2PO4 and 8.06 mM Na2HPO4, pH 7.4) according to the required DNA/peptide ratios
and concentrations and then were heated at 94°C for 8 min and allowed to re-anneal by
unplugging the heating block (slowly cooling to ambient temperature). All samples for
Peptide-DNA binding studies described in this chapter were prepared following this
procedure if not stated otherwise.
2.2.6. UV Job Plot Experiments
UV absorbance was monitored at 260 nm on UV-Vis HP 8543 with an external water
bath at 25 °C. Samples of varying molar ratios of peptide 1: DNA (dT10 or dT10C10T10)
were prepared by serial dilution while DNA concentration remained constant (7.5 µM for
dT10, 3.5 µM for dT10C10T10). All samples were annealed and preequilibrated in buffer as
previously described. Plots of A260 vs molar ratio were produced to determine the binding
stoichiometry of 1:2 or 1:1 respectively.
2.2.7. Fluorescence Job Plot (Quenching) Experiments
Fluorescence intensities were obtained on Perkin Elmer Luminescence Spectrometer LS
50B equipped with PTP 1 Temperature Programmer and water bath at 25 °C. Samples of
varying molar ratios of peptide 1: Flc-5’-dT10C10T10-3’-Dabcyl were prepared by serial
dilution while Flc-5’-dT10C10T10-3’-Dabcyl remained constant at 500 nM. All samples
were annealed and preequilibrated in buffer as previously described and were excited at
493 nm (excitation slit of 10 nm) and emission were recorded between 500-600 nm
45
(emmison slit of 10 nm) monitoring the maximum at 521 nm. The fluorescence intensity
vs molar ratio of peptide 1 was plotted to determine the binding stoichiometry of 1:1
between peptide 1 and Flc-5’-dT10C10T10-3’-Dabcyl.
2.2.8. Circular Dichroism (CD) Experiments
CD spectra were performed on Jasco J815 Circular Dichroism Spectrometer equipped
with Peltier device and water circulator. All measurements were taken at 25 °C in a
Hellma quartz cell (1 mm path length) from 350-210 nm at a scanning rate 50 nm/min,
data interval 0.5 nm, band width 1 nm and D.I.T 2 s. For each sample three scans were
collected, averaged and corrected for blanks. Total dN10 (T10, A10, C10) tracts
concentrations were remained at 10 µM in each binding reaction. Samples for CD
titration of dT10 with peptide 1 ranging from 0-20 µM were prepared by serial dilution.
Samples for CD binding study at a single DNA-Peptide ratio, 5 µM peptide 1 was used.
All samples were annealed and preequilibrated in buffer as previously described.
2.2.9. Thermal Denaturation Studies by UV
Thermal denaturation studies of triplex 5 and hairpin 6 were performed on Cary 100 Bio
UV-Visible Spectrophotometer (Varian) equipped with Cary Temperature Controller.
Samples were prepared in the binding buffer according to the required ratios and
concentrations. The single stands of DNA were mixed with peptide 1 to give final
concentrations of 2 µM dN10 (dT10 or dA10 or dC10) and 1 µM peptide 1 for triplex study;
2.5 µM dT10C10T10 and 2.5 µM peptide 1 for hairpin study; 1 µM, 2.5 µM and 3.75 µM
peptide 1 for self-structure study. All samples were annealed following general annealing
46
procedure. The temperature dependent absorbance of each sample was monitored from
10 to 90 °C at 260 nm with a ramp rate of 1 °C per minute and readings were taken every
0.5 °C increments. Tm values were determined from the first-order derivatives.
2.2.10. Thermal Denaturation Studies by Fluorescence
Fluorescence melting experiments for hairpin 6 were performed on Cary Eclipse
Fluorescence Spectrophotometer (Varian) equipped with Cary Temperature Controller.
The dual labeled dT10C10T10 contains fluorescein (Flc) as the fluorophore at 5’-end and
Dabcyl as the quencher at 3’-end. Freshly annealed solutions containing 250 nM Flc-5’-d
T10C10T10-3’-Dabcyl in the presence and absence of 250 nM peptide 1 were excited at
493 nm (excitation slit of 5 nm) and emission were recorded at 521 nm (emission slit of 5
nm) from 10 °C to 90 °C (upscan). The samples were then allowed to thermostat at 90 °C
for 5min followed by downscan from 90 °C to 10 °C. A 1 °C/min ramp rate with 0.5 °C
data interval was applied to both upscans and downscans. Tm values were determined
from the first-order derivatives.
2.2.11. Differential Scanning Calorimetry (DSC)
DSC was performed on Microcalorimeter VP-DSC. Peptide 1 in each sample was at 25
µM while dT10 and dT10C10T10 were 50 µM and 25 µM respectively. All samples were
annealed and preequilibrated as previously described and were thoroughly degassed by
ThermoVac. A 60 °C/h scan rate, 16 s filtering period and low feedback were applied to
both upscans (10 to 90 °C) and downscans (90 to 10 °C). Samples were allowed to
thermostate at 90 °C for 5 min before downscan started in each thermal cycle.
47
Background data was recorded by using the binding buffer in the sample cell and was
subtracted from other sample data. The final values of Cp were shown as the average of
six thermal cycles for each sample and were fitted to a Non-2-State binding model.
A
B
35
35
30
30
Cp (kcal/mole/°C)
Cp (kcal/mole/°C)
Model: MN2State
Tm: 47.50 ± 0.02 °C
H: -268. 60 ± 1.79 kcal/mole
Hv: 95.41 ± 0.78 kcal/mole
25
Model: MN2State
Tm: 48.67 ± 0.03 °C
H: 285. 70 ± 2.18 kcal/mole
Hv: 95.45 ± 0.90 kcal/mole
25
20
15
10
20
15
10
5
5
0
0
10
20
30
40
50
60
70
80
10
90
20
30
40
50
60
70
80
90
Temperature (°C)
Temperature (°C)
Figure 2.10. DSC traces of triplex 5 (black) with Non-2-State binding model fitted curves shown
in red. (A) Upscan and (B) Downscan.
48
B
60
60
50
50
Model: MN2State
Tm: 55.95 ± 0.01 °C
H: 319.90 ± 0.71 kcal/mole
Hv: 141.10 ± 0.39 kcal/mole
40
Cp (kcal/mole/°C)
Cp (kcal/mole/°C)
A
30
20
Model: MN2State
Tm: 54.62 ± 0.01 °C
H: -321.60 ± 1.23 kcal/mole
Hv: -139.00 ± 0.66 kcal/mole
40
30
20
10
10
0
0
10
20
30
40
50
60
70
80
10
90
20
30
40
50
60
70
80
90
Temperature (°C)
Temperature (°C)
Figure 2.11. DSC traces of Hairpin 6 (black) with Non-2-State binding model fitted curves
shown in red. (A) Upscan and (B) Downscan.
2.2.12. Fluorescence and Fluorescence Anisotropy Titration experiments were performed on Molecular Devices SpectraMax M5. Peptide 2
was used as the probe at a fixed amount 25 nM for Equilibrium Binding Assays and 500
nM (>>Kd) for Stoichiometric Binding Assays respectively. DNA oligomers as ligands
were serially diluted in binding buffer to desired concentrations and then an equal amount
from each stock was added into peptide 2 to give the final DNA-peptide 2 ratio for each
binding reaction. All samples were annealed and preequilibrated as previously described
before applying to 384 well, low volume, non-binding surface, round bottom, black
polystyrene Corning Assay Plate. Fluorescence and fluorescence anisotropy were
recorded at 525 nm upon excitation at 485 nm at 25 °C. All assays were performed in
49
triplicate and the values shown here were the averages from two separate experiments.
All experimental data were fitted by nonlinear regression analysis using LevenbergMarquardt algorithm in KaleidaGraph.
Equilibrium Binding Assays for Triplex 5.
(a)
A
B
Fluorescence Intensity (a. u.)
120
100
80
60
40
20
0
200
400
600
800
1000
1200
1400
Total dT10 (nM)
!!!!!!!
!
(b) Figure 2.12. Representative fitting data set for triplex 5: (A) Fluorescence intensity change as a
consequence of association reaction was monitored to obtain quenching factor Qb/Qf = 0.26
(where Qb and Qf is the quantum yield of bound and free fluorophore respectively). Qb/Qf from
this set
1 of data was applied to the calculation of fb (fraction bound) in analyzing anisotropy
values. (B) the Hill coefficient was added as a fitting variable fb = [dT10]n/(Kd+[dT10]n), yielding a
0.8
Fraction Bound
best fit with n=2.
0.6
0.4
Kd = 4064.6 ± 633.5 nM2
0.2
0
0
!!!!
!
!
!
200
400
600
[dT10] (nM)
800
1000
1200
!
50
- S24 -!
40
0
100
Equilibrium Binding Assays for Hairpin 6.
200
300
400
500
600
500
600
Total dT10C10T10 (nM)
A
B
120
28
Bound Peptide2 (nM)
Fluorescence Intensity (a. u.)
24
100
80
60
20
16
12
8
Kd = 2.7 ± 0.5 nM
4
40
0
100
200
300
400
500
0
600
Total dT10C10T10 (nM)
0
100
200
300
400
Total dT10C10T10 (nM)
!
Figure S17. Representative fitting data set for hairpin formation. (Top) F
a consequence
of association intensity
reaction was
monitored
Figure 2.13. Representative fitting data set for hairpin
6. (A) Fluorescence
change
as a to obtain quenchi
Bound peptide 2 concentration calculated from anisotropy values (Qb/Qf
dT10
T10 concentration
fittedQtob/Q
1:1f =
binding
model3,4: [Bound pepti
consequence
of association reaction was monitored
toC10
obtain
quenchingwas
factor
0.3. (B)
28
(sqrt((([RT]+[DNAT] +Kd)^ 2)-(4*[RT]*[DNAT]))))/2), where [RT] is the t
Bound24peptide 2 concentration calculated from anisotropy
values
0.3 applied)
vs10total
f =concentration
(25 nM), [DNA
theb/Q
total
of dT
C10T10 used in each bind
T] is(Q
Bound Peptide2 (nM)
dT10C10T10 concentration was fitted to 1:1 binding model83,84: [Bound
20
peptide 2] =
^
((([RT]+[DNAT]+Kd)-(sqrt((([RT]+[DNAT]+Kd) 2)-(4*[RT]*[DNAT]))))/2), where [RT] is the total
16
concentration of peptide 2 (25 nM), [DNAT] is the total concentration of dT10C10T10 used in each
12
binding reaction. R2 > 0.98.
8
Kd = 2.7 ± 0.5 nM
4
0
0
100
200
300
400
500
600
Total dT10C10T10 (nM)
2.2.13. Gel Mobility Shift Assay (carried out! by Yaowalak Pratumyot)
Figure S17. Representative fitting data set for hairpin formation. (Top) Fluorescence intensity change as
a consequence of association reaction was monitored to obtain quenching factor Qb/Qf= 0.3 (Bottom)
Bound
peptide
concentration calculated
anisotropy values
(Qb/Q
(a) applied)
vs total
f = 0.3 from
Triplex
5 2experiment
sample from
preparation:
20 nM
Cy5-dT
80 nM
dT10 were
10 and
dT10C10T10 concentration was fitted to 1:1 binding model3,4: [Bound peptide 2] = ((([RT]+[DNAT] +Kd)(sqrt((([RT]+[DNAT] +Kd)^ 2)-(4*[RT]*[DNAT]))))/2), where [RT] is the total concentration of peptide 2
2
incubated
increasing peptide 1 concnetration in 1X DPBS (Invitrogen)
with a final
(25
nM), [DNAwith
T] is the total concentration of dT10C10T10 used in each binding reaction. R >0.98.
loading volume of 10 µL. Samples were incubated at 94°C for 10 min
- S26and
-! then the
mixture was allowed to cool to R.T. over 2 hr. For hairpin 6 experiments: As above, with
20 nM Cy5-dT10C10T10 samples used in each well instead. After incubation, 2 µL of
loading buffer (80 % Glycerol) was added to all DNA-peptide 1 mixtures and 5 µL of
51
sample was loaded into the well of 22 % and 10% native polyacrylamide gel for dT10 and
dT10C10T10 respectively. The samples were subsequently electrophoresed on ice in an
electric field of 100 V using 1X TBE as a running buffer for 7 hours for dT10 and 2 hours
for dT10C10T10. After electrophoresis, Cy5 fluorescence gel images were acquired using a
Typhoon Trio Variable Mode Imager (Amersham Biosciences).
52
Chapter 3 Results and Discussion
53
3.1. Binding Stoichiometery
Nanoparticle assembly from melamine and cyanuric acid derivatives results from
noncovalent polymerization of the two-fold symmetric heterocycle recognition faces;
thymine has only one recognition face complementary to melamine, and thus discrete
assembly was anticipated. Gratifyingly, no large peptide-DNA aggregates were
detectable by dynamic light scattering, consistent with the model of discrete triplex
formation. Peptide-triggered base-stacking signatures were observed by UV absorbance
changes, with a 1:2 stoichiometry between 1 and dT10, consistent with bivalent
melamine-thymine recognition and formation of triplex 5 (Figure 2.1 and 3.1 A); peptide
1 alone did not induce such a signal (Figure 3.2).
Figure 3.1. Peptide 1 titrated into (A) dT10 and (B) Flc-dT10C10T10-Dabcyl, followed by UV
absorbance (260 nm) and fluorescein emission (521 nm), respectively. Saturation is observed at
33 and 50 mol% peptide, indicating 1:2 and 1:1 peptide: DNA binding stoichiometries in (A) and
(B), respectively.
54
Figure 3.2. UV absorbance (260nm) of peptide 1 alone at different concentrations.
Unlike PNA-DNA42 triplex structures which optimally form at high salt with divalent
metal ions (1-2 M NaCl, 50 mM MgCl2), robust assembly was observed under standard
salt conditions (Dulbecco’s phosphate buffered saline, DPBS), akin to the conditions
used by Eschenmoser and Krishnamurthy56 for PNA-DNA duplex formation.
Similarly, UV absorbance signatures indicated a 1:1 binding stoichiometry between 1 and
dT10C10T10 (Figure 3.4), as would be expected if an intramolecular peptide-DNA triplex
structure formed from the dT10 termini of dT10C10T10. Indeed, binding of 1 to FlcdT10C10T10-Dabcyl resulted in maximal fluorescein quenching at a 1:1 peptide-DNA
ratio, supportive of the formation of heterodimeric hairpin structure 6, which would bring
the 3’ and the 5’ end of the oligo in close proximity,56 resulting in efficient dabcyl
quenching of fluorescein (Figure 3.1 B).
55
0.9
0.7
A
260
0.8
0.6
50%
0.5
0
!!!!!!!!!!!!
20
40
60
80
Mole percent peptide 1
100
!
Figure S7. UV absorbance Job plot analysis of peptide 1 with dT10C10T10.
Figure 3.3. Peptide
1 titrated into dT10C10T10 followed by UV absorbance (260 nm). Saturation is
!
observed at 50 mol% peptide, indicating 1:1 peptide: DNA binding stoichiometry.
3.2. CD and Gel Mobility Shift
Triplex and hairpin formation was further corroborated by circular dichroism (CD),
which indicated significant structuring of DNA upon addition of peptide, signified by the
development of a negative CD signal at 260 nm at the expense of a positive CD signal at
280 nm, which we assign to the peptide complex and free DNA, respectively (Figure
3.4). Notably, while the peak at 280 nm is completely ablated in the triplex, there is a
residual peak in the hairpin; this is consistent with the presence of an unstructured C10
loop found in hairpin 6 but not triplex 5. Clean transformation of DNA to peptide-DNA
complex bands on native polyacrylamide electrophoresis indicated discrete peptide-DNA
recognition in both triplex and hairpin contexts (Figure 3.4).
56
- S14 -!
ciety
ciety
Communication
Communication
dimethylamine and
dimethylamine and
nd tetramethylated
nd tetramethylated
used in SPPS to
used in SPPS to
and cyanuric acid
and cyanuric
acid
erization
of the twoerization
of has
the only
twoes;
thymine
es; thymineand
has thus
only
melamine,
melamine,
andlarge
thus
tifyingly, no
atifyingly,
no light
large
e by dynamic
eofbydiscrete
dynamictriplex
light
discrete triplex
ngof signatures
were
ng
signatures
were
a 1:2 stoichiometry
a 1:2 stoichiometry
bivalent
melamine−
bivalent
melamine−
lex 5; peptide
alone
lex
5;
peptide
alone
and 2, Supporting
and 2, Supporting
Figure 3. Circular dichroism spectra in DPBS, pH 7.4, of (A) 1
□) vs dT10 spectra
alone (--)inand
(B) 1pH
complexed
complexed
dT10 (dichroism
Figure 3.with
Circular
DPBS,
7.4, of with
(A) 1
○
Figure 3.4.
Circular
dichroism
spectra
in
DPBS,
pH
7.4,
of
(A)
Peptide
1
complexed
with dT10
C
T
(
)
vs
dT
C
T
alone
(--).
Peptide
1
in
both
is
at
5
μM
dT
□
) vs10dT10 alone (--) and (B) 1 complexed with
complexed
10 10 10 with dT1010( 10
dTwith
T1010C10
are
at
concentration
(),
while
10C10
T10and
(○
) vsPeptide
dT10CdT
T1010and
alone
(--).
Peptide
in both
at10T510
dT
( ) vs dT
(B)
110complexed
dT
T101maintained
()
vs dTis
alone(--).
10C10(--)
10 alone
10C
10μM
and
5
μM,
respectively.
Electrophoretic
mobility
shift
assays
imaged
by
and
dT
C
T
are
maintained
at
10
concentration
(),
while
dT
10
10 1010
Peptide 1 in both is at 5µM concentration (—),
while dT
and10dT10C10T10 are maintained at 10
Cy5-dT
C10T10 by
Cy5
for (C) Electrophoretic
Cy5-dT10 (DNA
1) and (D)
andfluorescence
5 μM, respectively.
mobility
shift
assays10imaged
and 5 µM,
respectively.
Electrophoretic
mobility
shift
assays
imaged
by
Cy5
fluorescence
(DNA
in Cy5-dT
each lane,(DNA
with ) increasing
peptide 1 for (C)
2 ) at 20 nM
Cy5 fluorescence
for (C)
10
1 and (D) Cy5-dT10C10T10
from
left
right.(DNA
(E) 2lane,
Relative
electrophoretic
mobilities
Cy5-dT10concentration
(DNA1)2)andat(D)
Cy5-dT
) at 20 with
nM
in each
lane, with
increasing1peptide
10T10each
(DNA
20
nM10toCin
increasing
peptide
ofconcentration
the free DNA
oligos
and
their
peptide
complexes,
a
mixture
of
from
left
right. (E)
Relative electrophoretic
d (B) Flc-dT10C10T10-1 concentration from left to
right.
(E)toRelative
electrophoretic
mobilities of themobilities
free DNA oligos
the central
See SI for
further of
complex
5 and
DNAoligos
1 shown
of the free
DNA
andintheir
peptidelane.
complexes,
a mixture
fluorescein
peptide complexes, a mixture of complex 5 and DNA1 shown in the central lane.
dnm)
(B) and
Flc-dT
- theirdetails.
10C10T10and
complex 5 and DNA1 shown in the central lane. See SI for further
observed
and 50
nm) andat 33
fluorescein
details.
eptide:DNA
observed
at 33binding
and 50
eptide:DNA binding
ptide−DNA4 triplex
4 metal
t with divalent
triplex
ptide−DNA
obust
assembly
was
t with divalent metal
ulbecco’s
phosphate
obust
assembly
was
onditions
used
by
Dulbecco’s phosphate
PNA−DNA duplex
conditions used by
ures indicated a 1:1
PNA−DNA duplex
C10T10, as would be
tures
indicated a 1:1
NA triplex structure
C T10, as would be
T1010. Indeed,
binding
Cooperative melting was observed for triplex 5 and hairpin 6
by Cooperative
both UV and
fluorescence
dequenching
and5 and
differential
melting
was observed
for triplex
hairpin 6
scanning
calorimetry
(DSC)
(Figure
4,
SI).
The
hairpin
by both UV and fluorescence dequenching and differential
scanning calorimetry (DSC) (Figure 4, SI). The hairpin
57
3.3. Thermal Stability
Cooperative melting was observed for triplex 5 and hairpin 6 by UV, differential
scanning calorimetry (DSC) (Figure 3.5) and fluorescence dequenching (Figure 3.6). The
hairpin heterodimer structure 6 was more thermally stable (Tm = 54 °C) than the
heterotrimeric triplex 5 (Tm = 43 °C). For comparison, a dA10-T10 duplex has Tm = 35
°C, and a dA10- (T10)2 triplex has Tm = 17 °C in the presence of 50 mM MgCl2.85,86 It is
calculated that a dA10-T10 duplex (2 µM) will have Tm = 22.5 °C under similar salt
conditions (150 mM Na+, K+).87,88
Figure 3.5. (A) First-derivative plot of melting transitions of triplex 5 (--) and hairpin 6 (—)
followed by UV absorbance (260 nm). Normalized absorbance change is shown inset. (B) DSC
upscan traces of triplex 5 (—) and hairpin 6 (—), with downscan traces shown as dashed regular
and bold lines. Peptide-DNA ratios used in triplex 5 and hairpin 6 experiments were 1:2 and 1:1,
respectively. DSC and UV experiments were performed in DPBS, pH 7.4 at peptide
concentrations of 25 µM (DSC) and in UV experiments, 1 µM (5) and 2.5 µM (6).
58
F
50
0
10
20
30
40
50
60
70
80
90
Temperature (°C)
G. Fluorescence Melting Experiments
A
B
350
Flc-dT10C10T10-DABCYL
90°C to 0 °C
Upscan
15
Downscan
250
Flc-dT10C10T10-DABCYL
0°C to 90 °C
200
d(FL 521)/dT
Fluorescence Intensity (a. u.)
300
150
Hairpin duplex 6
90°C to 0 °C
Hairpin duplex 6
0°C to 90 °C
100
10
5
50
0
0
10
20
30
40
50
60
70
80
90
10
Temperature (°C)
20
30
40
50
60
70
80
90
Temperature (°C)
(Top)
Fluorescence
melting
curves for hairpin 6 (solid lines) and
Figure 3.6. (A) Fluorescence melting curves for Figure
hairpinS14.
6 (—)
and
free Flc-dT
10C10T10-Dabcyl (--
DAB (dotted lines); (Bottom) Corresponding first derivative curves for hairpin 6
); (B) Corresponding first derivative curves for hairpin 6. Fluorescence melting experiments were
Upscan
performed
in DPBS,
pH 7.4 at Flc-dT10C10T10-Dabcyl concentration of 250 nM in the presence or
15
Downscan
d(FL 521)/dT
absence of 250 nM peptide 1.
10
5
Notably, triplex 5 transition temperature yielded from DSC was slightly elevated (48 °C)
0
relative10 to20the30 other
while
the hairpin 6 Tm was very similar (55 °C). The
40
50 methods
60
70
80
90
Temperature (°C)
difference was likely due to the relatively higher complex concentrations used in DSC
Figure S14. (Top) Fluorescence melting curves for hairpin 6 (solid lines) and free Flc-5’-T10C10T10-3’DAB
(dotted lines); which
(Bottom) Corresponding
first stoichiometric
derivative curves for binding
hairpin 6. regime; consistent
experiments
were in the
with the
dissociation constants obtained from fluorescence and fluorescence anisotropy binding
isotherms (Figure 3.7).
- S21 -!
Reversible peptide-DNA complexation was supported by observation of endothermic
melting and exothermic cooling peaks by both fluorescence melt and DSC. Assembly of
complexes 5 and 6 was highly exothermic and reproducible over several thermal cycles
(ΔHtriplex = -285 kcal/mol peptide, ΔHhairpin = -314 kcal/mol), consistent with base59
stacking driven assembly.89-93 The large enthalpy values (-28.5 to -31.4 kcal/mol per
peptide: DNA triplet layer) are somewhat higher than, but similar to, enthalpies observed
for (TAT)n intramolecular DNA triplexes,94 which exhibit -21 kcal/mol per triplet stack.
Peptide-displayed melamine-DNA binding appears to have exothermic assembly profiles
and Tm values similar to those of DNA-DNA and melamine-cyanuric acid recognition,
suggestive of similar driving forces, perhaps to be expected given the similarity of
melamine and native nucleobases.
3.4. Binding Affinity
We quantified association using fluorescein-tagged peptide 2 binding to unlabeled DNA
(Figure 3.7). Complex formation was followed by both fluorescence anisotropy and
quenching, which accompanies assembly; similar results were obtained with the two
signatures. Using the stoichiometries established by titration (Figure 3.1), we fit the
binding curves to a trimer-monomer model to obtain an apparent Kd ≈ 4000 nM2 (by
quenching or anisotropy) for dissociation of triplex 5. A second transition was
undetectable by fitting to a two-step model or by observation of assembly processes,
suggesting that the peptide-dT10 heterodimer that forms initially must react rapidly with
another strand of dT10 to form the heterotrimer. Indeed, the coefficient derived from a
Hill-type binding isotherm was essentially 2, supportive of a cooperative 1:2 peptideDNA assembly process. A hypothetical equivalent two-step dissociation process with an
overall apparent Kd = 4000 nM2 would have Kd = 63 nM for both trimer-dimer and
dimer-monomer dissociation steps, which is the observed free DNA concentration at halfsaturation (Figure 3.7). A good fit to a 1:1 binding model83,84 was obtained from the
60
binding isotherm of peptide 2 to dT10C10T10, revealing an approximate Kd = 2.7 nM for
dissociation of hairpin 6.
Figure 3.7. Binding isotherms in DPBS, pH 7.4, of (A) peptide 2 binding to dT10 followed by
fluorescein quenching upon binding and (B) peptide 2 binding to dT10C10T10 followed by
fluorescence anisotropy. Solid lines show fits to (A) trimer-monomer 1:2 binding model
([fraction bound peptide 2] = [DNA]2/(Kd + [DNA]2), R2 ≥ 0.96) and (B) 1:1 binding model
(corrected
for
fluorescence
quenching):
[Bound
peptide
2]
=
((([RT]+[DNAT]+Kd)-
(sqrt((([RT]+[DNAT]+Kd)^2)-(4*[RT]*[DNAT]))))/2), where [RT] is the total concentration of
peptide 2 (25 nM), [DNAT] is the total concentration of dT10C10T10 used in each binding reaction.
R2 > 0.98.
3.5. Binding Selectivity
Given that peptides 1 and 2 and DNA are all anionic polyelectrolytes, we anticipate
negligible nonspecific electrostatic interactions. Indeed, partial and full methylation of
61
each melamine ring on the peptide to respectively yield peptides 3 and 4 abolished all
detectable binding to dT10 or dT10C10T10 (Figure 3.8). These observations are in
agreement with the critical role of the melamine recognition interface. As methylation
increases hydrophobicity, it is clear that assembly depends on recognition of donoracceptor patterning and not simply nonspecific hydrophobic collapse.
Notably, peptides 1 and 2 did not show any binding signatures when annealed with dA10,
dC10, and dG5A10, indicating a selectivity for thymine over the other native nucleobases
(Figure 3.9). Thus, melamine peptide recognition is relatively specific for the hydrogenbonding pattern of oligothymidine, as predicted. Taken together with the observed
stoichiometry of binding with dT10 and dT10C10T10, this is strongly supportive of the
expected bivalent thymine recognition by melamine (Figure 2.1).
62
Thermal denaturation studies and gel shift assays of control peptide 3 and peptide
dT10 were performed using the same procedures as for peptide 1.
Studies with control peptide 3 and peptide 4
(a)
Thermal denaturation studies and gel shift assays of control peptide 3 and peptide 4 with
dT10 were performed using the same procedures as for peptide 1.
(a)
A
1
dT10+Peptide3
dT10+Peptide4
dT10
0.8
Absorbance (260nm)
1
Absorbance (260nm)
0.8
0.6
(b)
0.4
0.4
0.2
0.2
0
0
dT10+Peptide3
dT10+Peptide4
dT10
0.6
10
20
30
50
60
40
50
60
70
80
Temperature (°C)
10
B
20
(b)
30
40
70
80
!
90
Temperature (°C)
90
!
!
Figure S20. (a) Absorbance (260 nm) vs Temperature plots for control peptide 3 and peptide 4
!
Figure 3.8.
(A) Absorbance
(260 nm)
vs
Temperature
for
control
peptide
andpeptide
4 dTPAGE
Figure
S20. Concentrations:
(a) Absorbance
(260
nm)
vs Temperature
plots
peptide
33and
with
2 uM
peptide
with 2plots
uM
dTfor
(b) Gel
shift assay
onpeptide
22%4 Native
for 5’
10.
10.control
Concentrations:
2 uM
peptide
with
2 uM dT
assay
on 22%
Native
PAGE
for 150,
5’-Cy5-dT
10. (b) Gel shift of
10
at
150
nM
with
increasing
concentrations
peptide
3
(0,
30,
50,
100,
225,
300,
750,
1
with dT10. Concentrations: 2 µM peptide with 2 µM dT10. (B) Electrophoretic mobility shift assay
at 150 nM with
of for
peptide
3 (0,430,
100, 150, 10225,
300,the
750,same
1200,condition
1500
nM)increasing
from leftconcentrations
to right. Study
peptide
and50,5’-Cy5-dT
under
yielded
left
to right.
for peptide
4 and
the same condition
yielded the
10 underconcentrations
imaged bynM)
Cy5from
fluorescence
forStudy
Cy5-dT
nM 5’-Cy5-dT
with increasing
of peptide
3 same
10 at 150
image.
image.
(0, 30, 50, 100, 150, 225, 300, 750, 1200, 1500 nM) from left to right. Study for peptide 4 and
Cy5-dT10 under the same condition yielded the similar result.
- S29 -!
63
peptide 1 for self-structure study. All samples were annealed following general annealing procedure. The
temperature dependent absorbance of each sample was monitored from 10 to 90°C at 260 nm with a ramp
rate of 1°C per minute and readings were taken
every 0.5°C increments. Tm values were determined from
(a)
the first-order derivatives.
(a)
(b)
A
5
0.12
dA10
Absorbance (260nm)
0.8
0.6
0.4
4
0.1
dC10+Peptide1
dC10
0.8
0.08
3
0.06
0.6
2
0.04
0.4
1
0.02
0.2
0.2
0
1
0
Anisotropy
Relative Fluorescence Intensity
dA10+Peptide1
1
Absorbance (260nm)
1
10
0
10
20
30
(a)
40
50
60
70
80
90
10
(b)
Temperature (°C)
20
30
0
1000
100
dG5A10 (nM)
40
50
60
70
80
90
Temperature (°C)
(c)
B
5
5
0.12
0.12
0.1
1uM Peptide1
2.5uM Peptide1
3.75uM Peptide1
0.15
0.08
3
0.06
0.1
2
0.04
0.05
1
0.02
0
0
1
10
10
20
30
40
60
70
80
0.1
0.08
3
0.06
2
0.04
1
0.02
0
0
1000
100
50
4
Anisotropy
Relative Fluorescence Intensity
4
Anisotropy
Relative
Fluorescence
Absorbance
(260nm) Intensity
0.2
1
10
100
0
1000
90
dC10 (nM)
dG5A10 (nM)
Temperature (°C)
(b)
Figure S13. Control experiments: absence of Figure
UV shift
melting.
Peptide()
1 and
with
(a) dA10(Δ)and
(b)for
with
continued
S18.or
Control
Fluorescence
Anisotropy
assays
binding selectivity s
dC10. (c) Thermal denaturation curves for peptide
1 alone
different
concentrations
to dC
rule
2 to thymine
overatother
nucleobases:
(a) dG5A10; (b)
No possible
change in signal is observe
10. out
secondary structures from peptide itself.
Figure 3.9. (A) Absorbance
(260 nm) vs Temperature plots for peptide 1 with control oligos dA10
5
Relative Fluorescence Intensity
0.12
and dC10. Absence of UV shift or melting signatures.
Concentrations: 1 µM peptide 1 with 2- S20
µM -!
4
0.1
dA10 or dC10. (B) Fluorescence () and Anisotropy
(Δ) assays for peptide 2 with control oligos
Anisotropy
0.08
dG5A10 and dC10.3 Peptide 2 concentration is constant
at 25 nM. (C) Circular dichroism spectra for
0.06
peptide 1 with control
oligos dA10 and dC10. Concentrations:
5 µM peptide 1 with 10 µM dA10 or
2
dC10.
0.04
1
0.02
0
1
10
100
0
1000
dC10 (nM)
Figure S18. Control Fluorescence () and Anisotropy (Δ) assays for binding selectivity study of peptide
2 to thymine over other nucleobases: (a) dG5A10; (b) dC10. No change in signal is observed.
64
- S27 -!
-10
220
240
260
280
300
320
340
Wavelength (nm)
Figure S9. CD spectra of other DNA oligomers with peptide 1. (a) free dC5T10C6/dG6T10G5 (T10 tracts 10
µM) and their peptide 1 complexes.
Figure 3.9 continued
6
6
4
4
CD (mdeg)
CD (mdeg)
C
2
0
0
dC10
dA10
-2
2
-2
dA10+Peptide1
dC10+Peptide1
Peptide1
Peptide1
-4
220
240
260
280
300
320
-4
340
Wavelength (nm)
220
240
260
280
300
Wavelength (nm)
320
340
!!!!!!!!!!!!!!!!!!!!
!
!
Figure S10. Control experiment: CD spectra of 10 µM dA10 with 5 µM
peptide
showingexperiment:
no change on
Figure
S11.1 Control
10 µM dC10 with 5 µM peptide 1.!
CD with peptide. (c) 10 µM dC10 with 5 µM peptide 1.
- S16 -!
65
Chapter 4
Conclusion
66
Overall, these data support the model of bivalent melamine-thymine recognition yielding
formation of novel discrete peptide-DNA triplex structures with robust binding affinities.
Notably, prior work from Eschenmoser and Krishnamurthy using similar diaminotriazine
nucleobase mimics derived from aspartic and glutamic acids resulted in duplex formation
with oligothymidines rather than triplex.54 Diaminotriazine has two exocyclic hydrogen
bond donor sites compared to three on melamine, which yields two potential thymine
recognition interfaces in melaminolysine and just one in diaminotriazine derivatives.
Thus, it appears that the assembly state pivots from dimer to trimer based on the addition
of a single hydrogen bond donor, with the increased entropic cost likely paid for by
increased base stacking. Polyvalent melamine-thymine recognition between peptides 1
and 2 with dT10 tracts is unique in that structure is induced from unstructured singlestranded oligothymidines to yield novel triplex and hairpin peptide-DNA hybrid
structures, thus expanding the range of non-native nucleobase-derived structures already
known.95,96 Though melamine recognition of preformed duplex with T-T/U-U mismatch
sites has been previously reported,67 the ability of polyvalent melamine peptides to broker
assembly with two non-interacting oligothymidine strands to form peptide-DNA triplex
structures is non-obvious.
We anticipate this novel approach can be employed to develop ligands for targeting long
stretches of mismatched bases that are present in important structural features such as
mutated sites of DNA and unpaired regions of RNA forming bulges and loops. The
melamine peptide we reported here in particular present promising artificial regulators to
manipulate the structure and function of thymine- and uracil-rich targets at DNA and
67
RNA level respectively. From a broader perspective, the designed element can serve as a
synthetic sequence-specific DNA binding domain (DBD) for artificial transcription
factors (ATFs) construction through modular design, which has become an essential
strategy toward development of genetic therapies.
High synthetic accessibility and the ease with which chemical functionality can be
introduced implied the high potential of our designed motif as a novel information
transfer system, such as a template to direct sequence-selective, nonenzymatic synthesis
of nucleic acid (or PNA) and peptide. Such investigations may gain insight into DNA
replication, transcription and translation in prebiotic chemistry.
As a model study of base pairing properties between polyvalent melamine and thymidine,
it will be important to gain more structural information of the peptide-DNA
heterotrimeric bundle motif. Of particular significance is to determine the crystal
structure. In addition, it also encourages follow-up investigations with respect to the trend
of diminishing DNA binding by varying the number of recognition element (M*).
Furthermore, to improve upon this lead melamine peptide design, chemical modifications
through peptide backbone to tune the binding affinity and specificity as well as to
understand in greater detail how such alterations might affect parameters such as cellular
uptake, nuclease resistance, tissue distribution and intracellular trafficking would be of
great interest before versatile in vivo activities can be fully accessed.
68
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76
Appendix A
Additional Characterization Data
77
annealed and preequilibrated in buffer as previously described. Plots of A260 vs molar ratio were produced
to determine the binding stoichiometry of 1:2 or 1:1.
0.7
0.65
0.6
A
260
0.55
0.5
0.45
Circular Dichroism Experiments.
0.4
CD spectra were performed on 33%
Jasco J815 Circular Dichroism Spectrometer equipped with Pel
0.35
and water circulator.
All measurements were taken at 25 °C in a Hellma quartz cell (1 mm p
from 350-210 nm at a scanning rate 50 nm/min, data interval 0.5 nm, band width 1 nm and D.I
0.3
each sample three 0scans were
collected,
averaged
for blanks. Total dN10 (T10
20
40
60
80 and corrected
100
tracts concentrations were remained at 10 uM in each binding reaction. Samples for CD titrati
Mole percent peptide 1
with peptide 1 ranging from 0-20
µM were prepared by serial dilution.
Samples for CD binding
!!!!!!!!!!!!!!!!!!!!
!
Figure
S6.
Additional
Job
plot
experiment
of
peptide
1
with
dT
.
10
DNA-Peptide
ratio,
5 µM
peptide1 1with
wasdTused.
All samples were annealed and preequi
Figure A1.single
Additional
UV Job plot
analysis
of peptide
10.
buffer as previously described.
3
2
CD (mdeg)
1
- S13 -!
0
-1
Peptide1 0 uM
-2
Peptide1 20 uM
-3
-4
220
!!!!!!!!!!!!
240
260
280
300
Wavelength (nm)
320
340
!
Figure A2.Figure
Additional
CD spectrum:
10 µM dT
of
S8. Additional
CDTitration
spectra:ofTitration
of 1010with
µMincreasing
dT10 withconcentration
increasing concentration
of pe
peptide 1.
78
peptide 1 for self-structure study. All samples were annealed following
temperature dependent absorbance of each sample was monitored from
rate of 1°C per minute and readings were taken every 0.5°C increments
the first-order derivatives.
(a)
(b)
dC5T10C6
dG6T10G5
dC5T10C6+dG6T10G5
dC5T10C6+Peptide1
dG6T10G5+Peptide1
dC5T10C6+dG6T10G5+Peptide1
Peptide1
10
1
1
dA10+Peptide1
5
dA10
0.8
Absorbance (260nm)
CD (mdeg)
Absorbance (260nm)
0.8
0
0.6
-5
0.4
-10
0.2 220
240
260
280
300
320
0.6
0.4
0.2
340
Wavelength (nm)
Figure S9. 0CD spectra of other DNA oligomers with peptide 1. (a) free dC5T100C6/dG6T10G5 (T10 t
10
20
30
40
10
20
30
40
50
60
70
80
90
and their
peptide
Figure A3. Additional µM)
CD spectrum:
free dC15complexes.
T10C6/dG6T10G5 (T10 tracts 10 µM) and their peptide
Tem
Temperature (°C)
1 complexes.
(c)
6
0.2
CD (mdeg)
Absorbance (260nm)
0.15
4
1uM Peptide1
2.5uM Peptide1
3.75uM Peptide1
2
0
0.1
dA10
-2
dA10+Peptide1
Peptide1
0.05
-4
220
240
260
280
300
320
340
Wavelength (nm)
!!!!!!!!!!!!!!!!!!!!0
!
Figure S10. Control experiment: CD spectra of 10 µM dA10 with 5 µM peptide 1 showing no ch
CD with peptide.
10 with
10
20(c) 10
30 µM
40 dC50
60 5 µM
70 peptide
80
90 1.
Temperature (°C)
Figure S13. Control experiments: absence of UV shift or melting. Pep
dC10. (c) Thermal denaturation curves for peptide 1 alone at different
Absence of UV meltingsecondary
signature rules
out possible
secondary
structures
-S
structures
from
peptide
itself.from peptide itself.
Figure A4. Additional thermal denaturation curve for peptide 1 alone at different concentrations.
79
Stoichiometric Binding Assays
0.13
1800
1600
0.11
0.1
(a)
1400
0.09
1200
0.08
1000
5
600
0
2
4
0.07
0.12
0.06
4
0.1
6
8
10
0.05
0.08
3
DNA/ Peptide2 Ratio
0.06
Anisotropy
Relative Fluorescence Intensity
800
Anisotropy
Fluorescence Intensity (a. u.)
0.12
Figure A5. Additional
stoichiometric
curves
for2 triplex
5 Cbf
or labeled
hairpin
6 by
titrating
Figure
S15. Detection of binding
triplex 5 or hairpin
6 formation
between
peptide
2 and
dT10 or 500 nM
peptide 2 with
dT10C10T10 by titration of 500 nM peptide 2 with dT10 or dT10C10T10. Both fluorescence (☐) and 0.04
anisotropy () were monitored for triplex 5. Only anisotropy was recorded for hairpin 6 (Δ).
dT10 or dT10C10T10. Both fluorescence ()
and anisotropy () were monitored
1
for
0.02
triplex 5. Only anisotropy was recorded for hairpin 6 (Δ).
0
1
10
0
1000
100
dT10 (nM)
(a)
(b)
A
B
5
2
0.14
0.1
0.06
2
0.04
1
0.02
0
1
10
100
0
1000
1.5
0.1
0.08
1
0.06
0.04
0.5
0.02
0
dT10 (nM)
0.12
Anisotropy
0.08
3
Relative Fluorescence Intensity
- S23 -!
4
Anisotropy
Relative Fluorescence Intensity
0.12
1
10
100
0
1000
dT10C10T10 (nM)
(b)
Figureand
S19.Anisotropy
Control Fluorescence
() and
(Δ)in
assays for free Cbf in
Figure A6. Additional control Fluorescence ()
(Δ) assays
forAnisotropy
free Cbf
dT10C10T10 (b) to rule out non-specific binding between the fluorophore and oligo
solution with (A) dT10 and (B) dT10C10T10 to rule out non-specific binding between the
2
0.14
0.12
1.5
0.1
0.08
1
0.06
0.5
0.04
0.02
0
0
80
Anisotropy
Relative Fluorescence Intensity
fluorophore and DNA oligomers.
Appendix B
1
H and 13C NMR Spectra of Amino Acid
Derivatives
81
Figure B1. 1H NMR of 1a.
Figure B2. 1H NMR of 1.
82
Figure B3. 1H NMR of 3a.
Figure B4. 1H NMR of 3b.
83
Figure B5. 1H NMR of 3c.
Figure B6. 1H NMR of 3.
84
Figure B7. 1H NMR of 4a.
Figure B8. 1H NMR of 4b.
85
Figure B9. 1H NMR of 4.
Figure B10. 13C NMR of 1a.
86
Figure B11. 13C NMR of 1.
Figure B12. 13C NMR of 3a.
87
Figure B13. 13C NMR of 3b.
Figure B14. 13C NMR of 3c.
88
Figure B15. 13C NMR of 3.
Figure B16. 13C NMR of 4a.
89
Figure B17. 13C NMR of 4b.
Figure B18. 13C NMR of 4.
90