“Spectroscopic study of natural and unnatural
derivatives of the pH-responsive cytosine-rich human
telomeric DNA for nanodevice insight”
— Bachelor thesis —
submitted to
department 14 (chemistry, biochemistry, pharmacy)
of the Johann-Wolfgang-von-Goethe University
Frankfurt am Main
in
June 2013
by
Katharina Holzhüter
First reviser:
Prof. Dr. Harald Schwalbe
Second Reviser: Prof. Dr. Clemens Glaubitz
Affirmation
Herewith I, Katharina Holzhüter, born on the 26th of February 1991 in Köln, declare that I have
written the here presented thesis on my own, without using any other than the cited sources and
mentioned auxiliary means. Everything that has been taken literally or analogously from other
publications is characterized as such. The same applies to the presented figures. This thesis has not
been presented, neither completely nor partly, to any examination board yet.
Frankfurt, 24th of June 2013
Terry Pratchett, “Feet of Clay”:
“People look down on stuff like geography and meteorology, and not only because they’re standing
on one and being soaked by the other. They don’t look quite like real science. † But geography is only
physics slowed down and with a few trees stuck on it, and meteorology is full of excitingly fashionable
chaos and complexity.”
†
That is to say, the sort you can use to give something three extra legs and then blow it up.
— List of contents —
List of contents
Affirmation .............................................................................................................................................. II
List of contents ....................................................................................................................................... IV
List of figures .......................................................................................................................................... VI
List of tables ......................................................................................................................................... VIII
List of abbreviations ............................................................................................................................... IX
1.
Introduction ..................................................................................................................... 1
2.
Theoretical Background ................................................................................................... 2
2.1.
Watson-Crick base pairings .............................................................................................. 2
2.1.1.
B-DNA ............................................................................................................................... 2
2.1.2.
A-DNA and Z-DNA............................................................................................................. 2
2.2.
Non Watson-Crick base pairings ...................................................................................... 3
2.2.1.
Triplex DNA....................................................................................................................... 3
2.2.2.
G-Quadruplex ................................................................................................................... 4
2.3.
i-motif ............................................................................................................................... 6
2.3.1.
Structure........................................................................................................................... 6
2.3.2.
Structural polymorphism ................................................................................................. 7
2.3.3.
Biological relevance.......................................................................................................... 7
2.3.4.
Competition between Watson-Crick double helix, G-Quadruplex and i-motif................ 8
2.4.
Nanodevices ..................................................................................................................... 8
2.5.
Circular dichroism spectroscopy ...................................................................................... 9
3.
Materials and methods .................................................................................................. 11
3.1.
Oligonucleotides............................................................................................................. 11
3.2.
DNA concentration measurements ................................................................................ 11
3.3.
Circular dichroism spectroscopy .................................................................................... 12
3.3.1.
Sample preparation ........................................................................................................ 12
3.3.2.
CD spectra measurements ............................................................................................. 13
3.3.3.
CD Melting curves .......................................................................................................... 13
3.4.
4.
Native gel electrophoresis.............................................................................................. 13
Results and discussion.................................................................................................... 15
4.1.
pH-response range of the c-rich structures ................................................................... 15
4.1.1.
I3 ..................................................................................................................................... 15
4.1.2.
I4 and I6: effect of the elongation of the cytosine-rich tracts ....................................... 20
4.1.3.
I3Me2 and I3Me4: effect of methylation on the 5-position of cytidine ........................ 29
4.1.4.
I3Br2 and I3Br4: effect of bromine substitution in the 5-position of cytidine............... 35
IV
— List of contents —
4.1.5.
4.2.
Interim Summary............................................................................................................ 40
Stoichiometry of the i-motif folding............................................................................... 41
4.2.1.
Native gel electrophoresis under acidic conditions (pH 5.0) ......................................... 42
4.2.2.
Native gel electrophoresis under basic conditions (pH 8.5) .......................................... 44
4.2.3.
Melting curves of I3Br2 at different concentrations. ..................................................... 45
4.3.
CD Melting curves: thermal stability of the i-motifs ...................................................... 46
4.3.1.
I3 ..................................................................................................................................... 48
4.3.2.
I4 and I6: effect of elongation of the cytosine-rich tracts on the thermal stability ....... 52
4.3.3.
I3Me2 and I3Me4: effect of 5N-methylation of cytidine on the thermal stability of imotifs ............................................................................................................................. 57
4.3.4.
I3Br2 and I3Br4: effect of 5N-bromination on the thermal stability of the i-motifs ..... 63
5.
Conclusion and outlook .................................................................................................. 67
5.1.
pH-response range ......................................................................................................... 67
5.2.
Stoichiometry of the i-motifs ......................................................................................... 68
5.3.
Thermal stability............................................................................................................. 68
6.
Zusammenfassung.......................................................................................................... 70
7.
Reference ....................................................................................................................... 75
V
— List of figures —
List of figures
Fig. 1: Schematic Illustration of a double-stranded oligonucleotide containing respectively two
guanosine-cytidine and thymidine-adenine canonical base pairings. .................................................... 2
Fig. 2: Comparison of Watson-Crick base pairs and Hoogsteen base pairs. ........................................... 3
Fig. 3: Guanosine tetrad, composed of 4 guanosines connected by eight Hoogsteen basepairs. .......... 4
Fig. 4: Scheme of the G-quadruplex formed by d(T2A(G3T2A)3) .............................................................. 5
Fig. 5: Schematic illustration of one hemiprotonated cytosine-cytosine base pair ................................ 6
Fig. 6: 5’E topology of d(C3TA2)C3
....................................................................................................... 7
Fig. 7: 3’E topology of d(C3TA2)C3 . ....................................................................................................... 7
Fig. 8: Schematic illustration of the origin of elliptical polarized light. ................................................... 9
Fig. 9: CD spectra of the first series of I3 (with 1.1 nmol DNA per sample). ......................................... 15
Fig. 10: CD spectra of the first measurements on I3 (with 3.6 nmol DNA per sample).. ...................... 16
Fig. 11: CD spectra of the second measurement series on I3 (with 3.6 nmol DNA per sample). ......... 16
Fig. 12: Plot of the molar ellipticity *deg∙cm²/dmol+ at 288 nm against pH for I3. ............................... 18
Fig. 13: CD spectra of the measurement series on I4 ........................................................................... 20
Fig. 14: Plot of the molar ellipticity [deg cm²/dmol] at 288 nm against pH for I4. ............................... 21
Fig. 15: Plot of the molar ellipticity [deg cm²/dmol] at 288 nm against pH for I4 with new values for
pH 5.6 to 6.0. ......................................................................................................................................... 22
Fig. 16: CD spectra of the measurement series on I6 (with 1.9 nmol DNA per sample). ...................... 24
Fig. 17: Plot of the molar ellipticity *deg∙cm²/dmol] at 288 nm against pH for I6 (1.9 nmol DNA per
sample). ................................................................................................................................................. 26
Fig. 18: Plot of the molar ellipticity *deg∙cm²/dmol+ at 288 nm against pH for I6 (0.3 nmol DNA per
sample). ................................................................................................................................................. 27
Fig. 19: Comparison of both pH response curves (molar ellipticity vs. pH) for both I6 measurement
series. .................................................................................................................................................... 27
Fig. 20: CD spectra of I3Me2. ................................................................................................................ 30
Fig. 21: CD spectra of I3Me4 ................................................................................................................. 30
Fig. 22: Plot of the molar ellipticity *deg∙cm²/dmol+ at 288 nm against pH for I3, I3Me2 and I3Me4. 33
Fig. 23: Schematic representation of a hemiprotonated C∙C+ base pair in DNA i-motifs...................... 35
Fig. 24: CD spectra of I3Br2 (with 1.1 nmol DNA per sample) .............................................................. 36
Fig. 25: CD spectra of I3Br4 (with 4.3 nmol DNA per sample). ............................................................. 36
Fig. 26: Plot of the molar ellipticity *deg∙cm²/dmol+ at 288 nm against pH for I3, I3Br2 and I3Br4. ... 38
Fig. 27: Native polyacrylamide gel (pH 5.0) for all sequences............................................................... 42
Fig. 28: Native polyacrylamide gel for I3 and I3Br2 under basic conditions (pH 8.5). .......................... 44
VI
— List of figures —
Fig. 29: Melting curves of I3Br2 with different concentrations (20 µM, 6,67 µM and 2 µM), all
measured at pH 5.4. .............................................................................................................................. 45
Fig. 30: Melting curves of I3 at pH 5.1 and pH 5.4. ............................................................................... 48
Fig. 31: Van’t Hoff plots (ln (K) vs. 1/T) for I3 at pH 5.1 and pH 5.4. ..................................................... 50
Fig. 32: Melting curves of I3, I4 and I6 at pH 5.1.. ................................................................................. 52
Fig. 33: Melting curves of I3, I4 and I6 at pH 5.4. .................................................................................. 53
Fig. 34: Van’t Hoff plots (ln (K) vs. 1/T) for I3, I4 and I6 at pH 5.1......................................................... 54
Fig. 35: Van’t Hoff plots (ln (K) vs. 1/T) for I3, I4 and I6 at pH 5.4......................................................... 54
Fig. 36: Melting curves of I3, I3Me2 and I3Me4 at pH 5.1. ................................................................... 57
Fig. 37: Melting curves of I3, I3Me2 and I3Me4 at pH 5.4.. .................................................................. 58
Fig. 38: Van’t Hoff plots (ln (K) vs. 1/T) for I3, I3Me2 and I3Me4 at pH 5.1. ......................................... 60
Fig. 39: Van’t Hoff plots (ln (K) vs. 1/T) for I3, I3Me2 and IeMe4 at pH 5.4. ......................................... 60
Fig. 40: Melting curves of I3, I3Br2 and I3Br4 at pH 5.1. ....................................................................... 63
Fig. 41: Melting curves of I3, I3Br2 and I3Br4 at pH 5.4. ....................................................................... 64
Fig. 42: Van’t Hoff plots (ln (K) vs. 1/T) for I3, I3Br2 and I3Br4 at pH 5.1. ............................................ 65
Fig. 43: Van’t Hoff plots (ln (K) vs. 1/T) for I3, I3Br2 and I3Br4 at pH 5.4. ............................................ 65
VII
— List of tables —
List of tables
Tab. 1: C-rich sequences analyzed in the presented work. ................................................................... 11
Tab. 2: Extinction coefficients that were used to calculate the concentrations of the oligonucleotides,
as well as assumed and calculated concentrations of the stock solutions and final CD sample
concentrations....................................................................................................................................... 12
Tab. 3: Fitting parameters for the I3 pH response curves. .................................................................... 19
Tab. 4: Fitting parameters for the two-state model fitting for both I4 data sets. ................................ 23
Tab. 5: Fitting parameters for the three-state model fitting for both I4 data sets. .............................. 23
Tab. 6: Fitting parameter for the pH response curves of the CD measurements on I3Me2 and I3Me4.
............................................................................................................................................................... 33
Tab. 7: Comparison of the maximum and minimum molar ellipticities of each sequence to that of I3
and ratio of maximum to minimum molar ellipticity for each sequence. ............................................ 37
Tab. 8: Fitting parameter for the pH response curves of the CD measurements on I3Br2 and I3Br4. . 38
Tab. 9: Summary of the results obtained from CD spectroscopy for all sequences. ............................ 40
Tab. 10: Fitting parameter for the two-state model fits for I3 at pH 5.1 and 5.4. ................................ 48
Tab. 11: Thermodynamic parameters calculated for I3 at pH 5,1 and 5,4............................................ 50
Tab. 12: Fitting parameter for the two-state model fits for I4 and I6 at pH 5.1 and 5.4. ..................... 53
Tab. 13: Calculated thermodynamic parameters for I3, I4 and I6 at pH 5,1 and 5,4. ........................... 55
Tab. 14: Fitting parameters for the two state model fits for I3Me2 and I3Me4 at pH 5.1 and 5-4. ..... 58
Tab. 15: Calculated thermodynamic parameters for I3, I3Me2 and I3Me4 from the melting curves
measured at pH 5.1 and 5.4. ................................................................................................................. 61
Tab. 16: Fitting parameters for the two state model fits for I3Br2 and I3Br4 at pH 5.1 and .,4. .......... 64
Tab. 17: Calculated thermodynamic parameters for I3, I3Br2 and I3Br4 from the melting curves
measured at pH 5.1 and 5.4. ................................................................................................................. 66
Tab. 18: C-reiche Sequenzen, die in dieser Arbeit untersucht wurden. ............................................... 70
Tab. 19: Zusammenfassung der Ergebnisse, die aus den CD-spektroskopischen Messungen bezüglich
der Stabilität gegenüber pH-Änderungen gewonnen wurden.............................................................. 71
Tab. 20: Zusammenfassung der thermodynamischen Parameter, die aus den Schmelzkurven
gewonnen wurden. ............................................................................................................................... 73
VIII
— List of abbreviations —
List of abbreviations
A
adenosine
C
cytosine
CD
Circular dichroism
d
deoxy
deg
degree
DNA
deoxyribonucleic acid
e.g.
for example
EDTA
ethylenediaminetetraacetate
et al.
et alii (“and others”)
Fig.
figure
G
guanosine
HPLC
high performance liquid chromatography
i.e.
that means
mdeg millidegree
N
nitrogen
NMR
nuclear magnetic resonance
R²
coefficient of determination
RNA
ribonucleic acid
T
thymidine
Tab.
table
Tm
melting temperature
Tris
Tris(hydroxymethyl)-aminomethane
ΔG
Gibbs energy
ΔH
enthalpy
ΔS
entropy
IX
— Introduction —
1.
Introduction
The here presented work deals with the i-motif, a special structure some oligonucleotides rich in
cytidine bases can adopt under certain circumstances. Of crucial importance for its occurrence is the
hemiprotonated cytosine-cytosine base pair (C∙C+), which was first described for crystals of acetyl
cytosine in 1962 by Marsh et al (Marsh et al. 1962). Because of that hemiprotonation, one of the
determining factors is the pH of the surroundings. Most of the observed sequences showed the imotif structure only under acidic conditions, although Zhuo et al. demonstrated in 2010 i-motif
formation under pH 7.0 and pH 7.1 for certain sequences (Zhou et al. 2010). The biological relevance
of the i-motif was doubted over a long period because of the acidic conditions it seems to require –
but further investigations led to the conclusion that it might play a role in biological systems, since crich structures have been found at various positions in the genomes of different organisms. Several
of the associated genes have been found related with cancer diseases, for example c-MYC, BCL-2,
RET, Rb and HIF-1α (Brazier et al. 2012). Researchers are trying to benefit from the pH dependency of
the i-motif as a H+-sensitive biosensor, used to monitor the pH in biological systems. For example,
Modi et al. designed such an i-motif based nanodevice (called the “I-Switch”) which is stable between
pH 5.5 and pH 6.8 and coupled to certain dyes that allow to monitor the folding status of the i-motif
via FRET (Modi et al. 2009). Sharma et al. invented an i-motif based pH switch (on the basis of the imotif induced aggregation of colloidal gold nanoparticles) which appears in two different colors
depending on the H+-concentration (red at pH 5.0, purple at pH 8.0) (Sharma et al. 2007). However,
few such nanodevices work around the physiological pH of 7.0, which would be of use to monitor pH
and pH changes in more diverse biological systems.
The presented work is based on a sequence adapted from the c-rich strand of the human telomere,
d(C3TA2)C3. Its i-motif forming capacity has been discussed several times (Leroy et al. 1994) (Mergny
1999) (Phan und Mergny 2002) (Zhou et al. 2010). Derivatives of this sequence are analyzed by CD
(circular dichroism) spectroscopy, regarding their pH response range and thermal stability with the
overall goal to find a sequence that may be used as a pH-sensitive nanodevice under physiological
conditions.
1
— Theoretical Background —
2.
Theoretical Background
2.1.
Watson-Crick base pairings
2.1.1. B-DNA
The structure of DNA molecules was first described by Watson and Crick in 1953. They proposed a
right-handed helical model in which two single strands, running in opposite directions, are coiled
around the same axis. The phosphate-and sugar-backbone is orientated to the outside, the bases are
pointing inside and are interacting via hydrogen bondings. These interactions are well-defined:
Adenine always forms a hydrogen bonding with thymine, guanosine forms one with cytosine
(Watson und Crick 1953). The resulting structure as reported by Watson and Crick is called “B-DNA”
nowadays and the base pairings as they are described here as “Watson-Crick base pairs” or
“canonical base pairs”. Fig. 1 shows a scheme of a double stranded oligonucleotide, each strand
consisting of four nucleotides, which is hold together by canonical base pairings.
Fig. 1: Schematic Illustration of a double-stranded oligonucleotide containing respectively two guanosinecytidine and thymidine-adenine canonical base pairings. The figure is taken from
https://en.wikipedia.org/wiki/File:DNA_chemical_structure.svg#file, 22-06-13 17:15
2.1.2. A-DNA and Z-DNA
Since the publication of the Watson-Crick DNA model, two other double stranded helical structures
stabilized by canonical base pairings have been discovered that are thought to be biologically active:
“A-DNA” and “Z-DNA”. Both are composed of canonical base pairings, but the overall shape is
different due to differing geometric properties, for example the degree of rotation round the helix
2
— Theoretical Background —
axis per base-pair, the inclination of base-pairs to the helix axis etc.. A-DNA is shorter and broader
than B-DNA and is said to be only present if the DNA is dehydrated (Basham et al. 1995). The lefthanded Z-DNA has a phosphate backbone with a zigzag pattern and is found when purine-pyrimidine
nucleotides alternate – the most favored form for a Z-conformation is (up to now) (GC)n. Z-DNA
forming sequences are mostly found in the telomeres of the human genome (Choi und Majima
2011). Interestingly, it was shown that B-DNA can be converted to Z-DNA by outer conditions as high
ionic strength, changes of solvent or bindings of proteins. This B-Z transition is thought to be useful
for designing nanodevices to monitor those conditions (Kwon und Rich 2005) (Rich und Zhang 2003).
2.2.
Non Watson-Crick base pairings
“Non Watson-Crick base pairs” or “non-canonical base pairs“ is a name for all base pairings between
nucleotides that are different to those Watson and Crick described in 1953. This includes Wobble
base pairs (often found between RNA nucleotides) and Hoogsteen base pairs, where the same bases
interact with each other as in Watson-Crick base pairs, but the geometry is different (Hoogsteen
1963). For a better visualization, Fig. 2 is given:
Fig. 2: Comparison of Watson-Crick base pairs and Hoogsteen base pairs. In both the same bases interact
with each other (AT and GC), but the purine is in the syn- instead of anti-conformation and therefore
different atoms participate in the hydrogen bonding. The figure is taken from
https://en.wikipedia.org/wiki/File:Hoogsteen_and_Watson%E2%80%93Crick_base_pairing.png, 22-06-13
17:32
Hoogsteen base pairings make the formation of triplex structures (section 2.2.1) and tetraplex
structures (G-quadruplex, section 2.2.2, and i-motif, section 2.3) possible (Choi und Majima 2011).
2.2.1. Triplex DNA
DNA triplexes are formed between three strands that each contain almost exclusive either purines or
pyrimidines. Two of the strands form a usual Watson-Crick base paired double helix; the third strand
binds via Hoogsteen base pairings to this B-DNA duplex. So the triplexes consist of either two
3
— Theoretical Background —
pyrimidine and one purine strand or of two purine and one pyrimidine strand (Frank-Kamenetskii
und Mirkin, 1995). Two types of triplexes are possible: the parallel motif and the antiparallel motif,
depending on the direction of the binding of the third strand. Triplexes consisting of one guanosine
strand and two cytosine strands are usually stabilized at low pH, because the cytosine is protonated
and an additional hydrogen bonding in the Hoogsteen base pairing is formed (Gilbert und Feigon
1999). Triplex formations have been used for gene targeting and recognition of specific DNA
sequences as well as for inhibition of gene activity, because the specifity of the third strand to the
homopurine-homopyrimidine double strand is high (Choi und Majima 2011).
2.2.2. G-Quadruplex
The G-quadruplex should be discussed in more detail here, since it is formed from G-rich strands that
usually are complementary to the C-rich strands that may fold into the i-motif.
Structure
Many G-rich sequences (DNA as well as RNA) have shown to form specific structures under
physiological conditions. These structures base on π-π stacking interactions between planar tetrads
of guanosines. The four guanosines for their part are bound to each other by eight Hoogsteen base
pairings (Choi und Majima 2011). Such a guanosine tetrad is shown in Fig. 3.
Fig. 3: Guanosine tetrad, composed of 4 guanosines connected by eight Hoogsteen basepairs. The figure is
taken from Sen und Gilbert (1988).
4
— Theoretical Background —
A typical G-quadruplex, formed from the sequence d(T2A(G3T2A)3 of the human telomere, can be
seen in Fig. 4.The guanosines are bound in the tetrads, connected by TTA-loops.
Fig. 4: Scheme of the G-quadruplex formed by d(T2A(G3T2A)3). The dotted lines correspond to the hydrogen
bondings that connect the four guanosines of each tetrad. The figure is taken from Sundquist und Klug
(1989).
G-quadruplexes show very high structural polymorphism, i.e. they can adopt many different
structures depending on various factors, such as nucleotide sequence, length of the g-tracts, loop
length, orientation of the strands. Mono- and bivalent cations which coordinate into the G-tetrads
are able to stabilize G-quadruplexes. Besides, they can be formed tetra-, bi- or monomolecularly
(Burge et al. 2006) (Choi und Majima 2011). Trent et al. observed at least eight possible
monomeric
quadruplex
structures
formed
by
the
same
sequence,
d(G2TG2TG2TG2T2GTG2TG2TG2TG2), by NMR and chromatography (Dailey et al. 2010). Because of the
stabilizing effect of cations, G-quadruplexes are used as electrical nanoswitches, for example
triggered by an increase in K+-concentration (Choi und Majima 2011).
Biological relevance
G-rich sequences can be found not only in the human telomeres, but also in many gene promoter
regions and in the non-coding regions of genes. The vertebrate telomeres are special about this,
because here a single stranded g-rich overhang is existing with a length of about 150 nucleotides. Up
to six g-quadruplexes can be formed here one after another. Besides the single stranded overhang,
the telomeres are consisting of double-helices where a g-rich strand interacts with a complementary
c-rich strand. Several ex-vivo-experiments show evidence for many different g-quadruplex structures
that may be formed under conditions occuring also in living systems (Burge et al. 2006). Several small
molecules were found to bind and induce the folding of the 3’-single strand into G-quadruplexes,
which inhibits the telomerase. Stabilizing the Q-quadruplexes is therefore tested as a anticancer
strategy (Balasubramanian et al. 2011).
In gene promoter regions it was proven that g-quadruplex stabilization is able to suppress the c-myc
transcriptional activation. The c-myc gene encodes the c-myc protein which enhances the expression
5
— Theoretical Background —
of other genes. Q-guadruplexes prevent the NM23H2 transcriptional activation protein from binding
to the nuclease hypersensitive element III1 sequence of the MYC promoter. G-quadruplex formation
was also shown for the human VEGF (vascular endothelial growth factor) gene as well as for the
genes of BCL-2 (B-cell lymphoma 2), KIT (coding for the stem cell growth factor) and MYB (coding for
the Myb proto-oncogene). All of them are associated with cancer diseases and researchers are
therefore trying to find ways to regulate their activity via g-quadruplex formation and stabilization
(Balasubramanian et al. 2011).
2.3.
i-motif
2.3.1. Structure
The first i-motif structure was observed for d(TC5) in 1993. Gehring and coworkers showed by NMR
spectroscopy that under acidic conditions a four-stranded complex is formed. Two strands each form
base paired parallel duplexes, whereas the two duplexes associate in an antiparallel manner. The
base pairs in this four-stranded complex are intercalated with each other, and the sugar-phosphate
backbones interact pairwise over van-der-Waals forces. Essentially important for the formation of
this tetrad is the hemiprotonated cytosine-cytosine base pair (C∙C+). In the tetramer of d(TC5), ten of
them are formed (Gehring et al. 1993). A scheme of one single C∙C+ base pairing is shown in Fig. 5.
Fig. 5: Schematic illustration of one hemiprotonated cytosine-cytosine base pair. The three hydrogen
bonds that connect the bases are shown with dotted lines. The figure is taken from Gehring et al. (1993).
According to Gehring et al., the tetraplex has several energetic advantages over the formation of two
duplexes: there are 11 base stacking interactions in the tetrad, but only 10 in two duplexes. The
N3-nitrogens of the cytosines are more separated from each other in a tetrad than in a duplex. The
carbonyl and amide dipoles in successive base pairs are reversed orientated, which leads to favorable
electrostatic interactions. The formerly mentioned sugar-sugar contacts are also an energetic
advantage that would be absent in duplexes. Up to now, the i-motif is the only known structure for
nucleic acids that involves this form of base intercalations (Guéron und Leroy 2000).
6
— Theoretical Background —
2.3.2. Structural polymorphism
The i-motif can be formed, as described, by four single-stranded c-rich sequences; also the formation
by dimers containing two cytidine-stretches as well as intramolecular foldings of sequences with four
stretches of cytosines are possible. In the last two cases, the c-stretches usually are separated by an
intermediate linker. In general, two different i-motif structures are possible which have different
intercalation topologies, termed 3’E and 5’E; in the 3’E topology, the outer cytidine is on the 3’end, in
the 5’E it is located at the 5’end of the stretch. Schematic illustration of these both topologies in the
i-motif structure of d(C3TA2)C3, a fragment of the human telomere, are shown in Fig. 6 and Fig 7.
Fig. 6: 5’E topology of d(C3TA2)C3.
Fig. 7: 3’E topology of d(C3TA2)C3.
Both figures are taken from Lieblein et al. (2012).
Lieblein et al. proposed for this sequence at equilibrium the 5’E topology as the major conformation,
the 3’E conformation as the minor conformation (Lieblein et al. 2012). Which topology is favored is
sequence-dependent. The topology of the loops may also vary, depending on anti- or syn-orientation
of the looping bases (Guéron und Leroy 2000). However, it is evident that i-motifs reveal no such
great structural diversity as g-quadruplexes.
2.3.3. Biological relevance
The biological relevance of the i-motif was doubted over a long time because it was only found under
acidic conditions. Cytosine-rich sequences can not only be found in the vertebrate telomere, but also
in various gene promoters, and over the last years it has been shown for many of them that i-motif
formation may be possible under physiological conditions. For example, Brazier et al. showed i-motif
formation for the gene promoters of HIF-1α, h-tert and PDGF-A close to physiological temperature
and pH (Brazier et al. 2012). For others, it is shown that they are able to fold in vivo into an i-motif
under acidic conditions, for example for the proximal promoter sequence of the gene for the
vascular endothial growth factor (Guo et al. 2008) or in retinoblastoma susceptibility genes (Xu
7
— Theoretical Background —
2006). Conditions under which the i-motif formation occurs under physiological conditions are
considerable, especially because living organisms contain acid compartments, for example the
lysosome, the interior part of endocytic vesicles or parts of the Golgi apparatus (Anderson und Orci
1988). Besides that, proteins have been discovered that recognize c-rich sequences which are also
capable of i-motif formation (Eid und Sollner-Webb 1995) (Sarig et al. 1997). Finally, Zhuo et al.
demonstrated a distinct population of fully folded i-motif structures for some cytosine-rich structures
at pH 7.0 and 4°C (Zhou et al. 2010).
2.3.4. Competition between Watson-Crick double helix, G-Quadruplex and i-motif
Most cytosine-rich DNA sequences are found in double helical chromosomes. Whether i-motif
formation occurs in vivo or not is therefore determined by the equilibrium between double helix and
single-stranded DNA. Phan and Mergny showed for sequences from the human telomeric DNA the
presence of all three possible structures, Watson-Crick double helix, G-quadruplex and i-motif, in
equilibrium at pH 4.5. They derived from this observation that at low pHs the double strand
dissociates into two single strands which form i-motif- and g-quadruplex structures. They proposed
that under physiological conditions the double-helix was favored, but the formation of i-motif and gquadruplex may be stabilized under certain conditions (high temperature, low pH, intermolecular
interactions with other molecules, superhelical stress). Interestingly, the formation of the Gquadruplex was not a competitor to the double helix; its formation did not occur until the pH favored
the i-motif formation (Phan und Mergny 2002). Additionaly, Li et al. observed a stabilization of the Gquadruplex in competition with the double strand by certain cation species that led to dissociation
even at higher pH values (Li et al. 2002).
2.4.
Nanodevices
Nanodevices are, said in simplified words, very small useful tools that can be applied to living
systems to deliver information to the researcher. DNA is extremely useful for nanometer scale
applications because of its ability to self-assemble (Alberti und Mergny 2003). I-motif based
nanodevices are usually used to monitor pH changes because of the pH dependency of the C∙C+ base
pair. Liu et al., for example, attached a c-rich sequence, coupled to a rhodamine green fluorophore at
the 3’ end, via a -C6SH linker to a thin-gold covered surface. Is the pH lowered, the c-rich structure
folds into the i-motif and the fluorophore is brought into proximity to the gold surface. The dye’s
fluorescence is quenched by energy transfer from the fluorophore to the gold surface. If the pH is
changed to more basic conditions, the i-motif unfolds, the fluorophore moves away from the gold
surface and its fluorescence is restored (Liu et al. 2006). Xu et al. invented a device, based on i-motif,
g-quadruplex and the GC-Watson-Crick duplex, that is able to bind and release the telomere-binding
protein TRF-1 and other small g-quadruplex binding molecules. This is interesting for example to
control the concentration of one of these small molecules in the cell (Xu et al. 2007). However, few i8
— Theoretical Background —
motif based applications are accessible yet that work at more basic conditions making it possible to
monitor cellular changes that are induced by pH shifts away from the acidic pH range. To observe
how certain sequence alterations influence the pH response range of a known i-motif forming
sequence was the overall aim of the work that is presented here.
2.5.
Circular dichroism spectroscopy
Circular dichroism spectroscopy is a technique that is sensitive to certain structural transformations
(Kelly et al. 2005). Circular dichroism is a phenomenon of optical activity and therefore a property of
all molecules that have at least one asymmetric group of atoms (Beychok 1966). It results from the
different extent of absorption of left- and right-circularly polarized light. Each light beam of plane
polarized light can be imagined as consisting of two light beams that are circularly polarized, the one
clockwise (right handed), and the other counter-clockwise (left-handed). A material that shows
circular dichroism absorbs both circularly polarized light beams to a different extent; the result is an
elliptical polarization (Fig. 8).
Fig. 8: Schematic illustration of the origin of elliptical polarized light. The right-handed circularly polarized
light is absorbed to a greater extent than the left-handed circularly polarized light. The result is an
elliptical polarized light that is rotated by the angle α to the original direction of propagation. The figure
is taken from Beychok (1966).
How elliptical the plane-polarized light gets after it has passed the material is dependent on the
difference in the absorption of the differently circularly polarized light beams. A CD
spectropolarimeter measures the difference in absorbance between the left- and the right-circularly
polarized light beam. This is then converted into ellipticity units (θ). The ellipticity is defined as the
arc tangent of the quotient of B’ and A’, the minor and major axes of the ellipse. The relationship
between absorption difference and ellipticity is given by θ = 32,89 ΔA [deg] (Kelly et al. 2005).
9
— Theoretical Background —
CD spectroscopy is extremely useful for analyzing i-motif structures, because these show a
characteristic CD spectrum that has been reported frequently: i-motifs present a positive ellipticity
band between 285 nm and 290 nm and a negative ellipticity band between 260 and 275 nm (Manzini
et al. 1994) (Mergny et al. 1995) (Simonsson et al. 2000) (Guo et al. 2007) (Kendrick et al. 2009) (Zhou
et al. 2010) (Dembska et al. 2013)
10
— Materials and methods —
3.
Materials and methods
3.1.
Oligonucleotides
Spectroscopic studies were performed on the sequences presented in the table below (Tab. 1).
Tab. 1: C-rich sequences analyzed in the presented work.
name
Sequence (5’-3’)
I3
CCC TAA CCC TAA CCC TAA CCC
I4
CCC CTA ACC CCT AAC CCC TAA CCC C
I6
CCC CCC TAA CCC CCC TAA CCC CCC TAA CCC CCC
I3Me2
CCC TAA C5mCC TAA CCC TAA C5mCC
I3Me4
C5mCC TAA C5mCC TAA C5mCC TAA C5mCC
I3Br2
CCC TAA C5BrCC TAA CCC TAA C5BrCC TAA
I3Br4
C5BrCC TAA C5BrCC TAA C5BrCC TAA C5BrCC
I3, I4, I6, I3Me2 and I3Me4 were purchased from Eurofins MWG Operon (D-Ebersberg) in 0.2 µmol
scale, except I3 which was ordered in a 10 µmol scale (for supplementary applications not correlated
to the project described herein), with a HPLC purification. The I3 sample was further purified by Elke
Stirnal, freeze-dried and desalted using microconcentrators Vivaspin 2 mL (MWCO 3000) (Sartorus,
D-Göttingen). I3Br2 and I3Br4 oligonucleotides were synthesized by iba – solutions for life science (DGöttingen), the former in 1.2 µmol and the latter in 0.2 µmol scale, with a HPLC purification as well.
I4, I6, I3Me2 and I3Me4 were delivered lyophilized. According to the mole quantities indicated by the
provider the samples were dissolved in an appropriate ddH2O volume to reach a concentration of
0.72 mM. I3Br2 and I3Br4 were received already in solution. For more convenience they were freezedried and dissolved in an appropriate ddH2O volume to reach the concentration of 0.72 mM.
3.2.
DNA concentration measurements
The first CD measurement on I4 revealed that the concentration of the stock solution was lower than
expected. The concentrations were then checked for all the oligonucleotides by UV/Vis-spectroscopy.
The optical density was measured on samples with a supposed concentration of 2.4 µM on a
Cary50 UV/Vis-spectrophotometer (Varian). The samples were measured in a quartz cuvette of 1 cm
path length. The baseline correction was performed on a ddH2O solution. The concentrations of the
oligonucleotides were calculated using the Lambert-Beer law (equation 1):
𝐸𝜆 = 𝑙𝑔
𝐼0
𝐼
= 𝜀𝜆 ∙ 𝑐 ∙ 𝑑
(equation 1)
In this equation, Eλ is the extinction of the material, which can be described by the logarithm of the
quotient of I0 (intensity of the irradiated light) and I (intensity of the light that has passed the
11
— Materials and methods —
material) or as product of the extinction coefficient ελ (in m²/mol), the concentration c (in mol/L) and
the path length (in m).
The extinction coefficients used are listed in Tab. 2. Regarding I4 and I6, the nearest-neighbor
method of Cantor et al. was applied to obtain this parameter (Cantor et al. 1970). For I3Br2 and
I3Br4, the extinction coefficients given on the synthesis report were used. For the methyl-modified
sequences the extinction coefficients were not provided by the supplier, so the extinction
coefficients were determined by using a base composition method (summing up the individual
extinction coefficients of each base multiplied by the number of its occurrence). For 5-Me-dC a ε260 of
5.7 mL/µmol was used1. The results of the calculations are summarized in Tab. 2.
Tab. 2: Extinction coefficients that were used to calculate the concentrations of the oligonucleotides, as well as assumed
and calculated concentrations of the stock solutions and final CD sample concentrations.
name
extinction coefficient
Assumed
Calculated
Concentration
[M-1 cm-1]
concentration [mM]
concentration from
of DNA per CD
OD [mM]
sample [µM]
I3
185900
2.03
0.27
6.3
I3
185900
2.56
2.56
20
I4
214700
0.72
0.63
17.5
I4
214700
0.72
0.57
15.8
I6
272300
0.72
0.38
10.6
I6
272300
0.72
0.07
1.9
I3Me2
183510
0.72
0.56
15.6
I3Me4
180450
0.72
0.69
19.2
I3Br2
173250
0.72
0.38
10.6
I3Br4
159930
0.72
0.86
23.9
3.3.
Circular dichroism spectroscopy
3.3.1. Sample preparation
To analyze the pH dependency of the i-motif folding, phosphate buffers (made from monopotassium
phosphate (KH2PO4) and dipotassium phosphate (K2HPO4) solutions) with pH values between 4.8
and 7.0 were established. For each sample, the buffer concentration was 400 mM, whereas the
concentration of the individual DNA sequences varied between 1.9 µM and 20 µM (see section 3.2).
Buffer and sample pH values were confirmed by measurements with a Schott Instruments Lab850
pH-meter equipped with a Hamilton pH Minitrode. All samples were heated to 95°C for 5 minutes
1
http://www.glenresearch.com/Technical/Extinctions.html
12
— Materials and methods —
and stored on ice for 15 minutes before any experiment to solve weakly bound secondary structures
and to guarantee the comparableness of the samples.
3.3.2. CD spectra measurements
The CD spectra and melting curves were recorded on a Jasco J-810 CD spectropolarimeter equipped
with a Jasco PTC-4235/L Peltier thermostated cell holder. The cell chamber was flushed with a
constant nitrogen flow to avoid water condensation on the measurement cuvette. For all
measurements, the same CD quartz cuvette with a path length of 1 mm was used.
The CD spectra were recorded at 25°C over a wavelength range from 220 nm to 330 nm (data pitch
0.2 nm). The spectra are the result of the average of the accumulation of three records; for all pH
values a baseline correction was done using a buffer solution.
The obtained ellipticity values were transformed into molar ellipticities according to equation 2:
𝜃 =
𝜃 ∙𝑀
𝑐 ∙𝑙 ∙10
(equation 2)
[θ] is the molar ellipticity in deg∙cm²∙dmol-1, θ is the ellipticity in mdeg, M the molar weight in g∙mol-1,
l the path length in cm and c the concentration of the sample in g∙ml-1. As the path length of the
cuvette used is l = 0,1 cm, equation 2 can be converted to equation 3 with θ in mdeg and c in mol∙L-1.
θ =
θ
c
(equation 3)
3.3.3. CD Melting curves
Melting curves were generated by monitoring the ellipticity at 288 nm along a temperature gradient
from 4°C to 95°C with a heating rate of 0.5°C/min. Melting curves were on the one hand recorded for
pH 5.1 for all sequences, on the other hand for pH 5.4. If possible, the samples from the CD spectra
records were re-used.
Again the ellipticities were commuted into molar ellipticities and normalized using the following
equation:
𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑠𝑒𝑑 𝑣𝑎𝑙𝑢𝑒 =
(𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 𝑣𝑎𝑙𝑢𝑒 − 𝑙𝑜𝑤𝑒𝑠𝑡 𝑣𝑎𝑙𝑢𝑒)/(ℎ𝑖𝑔ℎ𝑒𝑠𝑡 𝑣𝑎𝑙𝑢𝑒 − 𝑙𝑜𝑤𝑒𝑠𝑡 𝑣𝑎𝑙𝑢𝑒)
(equation 4)
Normalization makes it possible to compare the melting of the individual DNA sequences with each
other, because not the absolute molar ellipticities are treated but the ratio of folded and unfolded
molecules.
3.4.
Native gel electrophoresis
A native gel electrophoresis was performed, using a 20% polyacrylamide gel which pH was set to 5.0
with TAE buffer (Tris, acetic acid, EDTA) used as running buffer as well. 3.5 nmol of each
13
— Materials and methods —
oligonucleotide were mixed with TAE buffer and loading buffer containing glycerol (87%) and xylene
cyanol FF (0.1%) as dye. The samples were heated to 95°C for 5 minutes and then cooled on ice for
15 minutes before loading on the gel. As size marker a 2:1 mixture of I3 and its G-rich complement
was used. A voltage of 60 V was applied and the gel ran for approximately 4 hours at room
temperature with water cooling. DNA bands were visualized on a white background by UVshadowing and photographed with a digital camera.
I3Br2 was further analyzed with a gel electrophoresis under similar conditions, but this time with a
TAE buffer that pH was set to pH 8.5. I3 was used as a size marker.
14
— Results and discussion —
4.
Results and discussion
4.1.
pH-response range of the c-rich structures
Folding of the i-motif structure can be observed by using CD spectroscopy, since the i-motif shows a
very characteristic spectrum which has been reported for c-rich structures in acid conditions various
times: the CD spectra of i-motif structures are known to present a positive band between 285 and
290 nm and a negative band between 260 and 275 nm (Simonsson et al. 2000) (Guo et al. 2007)
(Kendrick et al. 2009). In the beginning, all sequences were analyzed by CD spectroscopy to proof
their i-motif folding capacity and determine their pH-response range.
4.1.1. I3
First of all, the I3 sequence was analyzed that has been chosen as a reference for the other
sequences since the other sequences are derived from it.
CD spectra
CD measurements were first performed on a series of I3 where each sample contained 6.3 µM DNA
(1.1 nmol). This low concentration was not intentional (see for section 3.2 DNA concentration
measurements). Later two series were measured with 20 µM DNA/3.6 nmol per sample. The CD
spectra obtained for the three series of measurements for I3 and the pH response curves are shown
in Fig. 9 to Fig. 11.
Fig. 9: CD spectra of the first series of I3 (with 1.1 nmol DNA per sample). The ellipticity [mdeg] in a
wavelength range of 220 to 330 nm at different pH (5.0 – 7.0) was measured and converted into molar
ellipticity *deg∙cm²/dmol+. The wavelengths that indicate the presence of i-motif are marked by blue lines,
the wavelengths that indicate the unfolded structure by green ones. The isosbestic points are either
marked by one of these or by a dashed line.
15
— Results and discussion —
Fig. 10: CD spectra of the first measurements on I3 (with 3.6 nmol DNA per sample). The ellipticity [mdeg]
in a wavelength range of 220 to 330 nm at different pH (5.1 - 7.0) was measured and converted into molar
ellipticity *deg∙cm²/dmol+. The wavelengths that indicate the presence of i-motif are marked by blue lines,
the wavelengths that indicate the unfolded structure by green ones. The isosbestic points are either
marked by one of these or by a dashed line.
Fig. 11: CD spectra of the second measurement series on I3 (with 3.6 nmol DNA per sample). The ellipticity
[mdeg] in a wavelength range of 220 to 330 nm at different pH (5.1 – 7.0) was measured and converted
into molar ellipticity *deg∙cm²/dmol+. The wavelengths that indicate the presence of i-motif are marked by
blue lines, the wavelengths that indicate the unfolded structure by green ones. The isosbestic points are
either marked by one of these or by a dashed line.
The curves show for pH values lower than 6.0 a positive band at 288 nm and a negative one at about
257 nm, which is typical for the i-motif structure. For pH values beyond 6.0 the intensity maxima can
in contrast be observed at 273 nm and 247 nm which corresponds to a single-stranded DNA coil
(Brahms und Mommaerts, W. F. H. M 1964) (Beychok 1966). The decrease of the ellipticities intensity
at 288 and 257 nm and the shift of the bands towards lower wavelengths show partial folded states
of the sample due to the reduced H+-concentration. For all the measurements the transition
16
— Results and discussion —
midpoint of the unfolding (where the ratio of folded and unfolded molecules equals 1) seems to be
around pH 6.0.
The spectra of the first series, the one with only 1.1 nmol DNA per sample, show high noise due to
the low DNA concentration. The curves at the individual pH values seem to have isosbestic points at
247 and 277.5 nm, i.e. points where the spectra cross each other. An isosbestic point occurs, when all
components in the solution have the same absorptivity at this particular wavelength (if the overall
concentration stays the same) (Cohen und Fischer 1962). Normally this can be taken as a hint for the
presence of only two different species in the solution (in this case a folded and an unfolded species)
as it is very unlikely that a third species is generated that has the very same absorptivity at the same
wavelength as the other two (Berlett et al. 2000). The formation of a population with no absorptivity
at all (which would not affect the occurrence of an isosbestic point) can be excluded as DNA in all
cases reported so far shows optical activity (Beychok 1966) (Brahms und Mommaerts, W. F. H. M
1964). So the observed spectra deliver evidence that the unfolding of the i-motif takes course as
expected.
The spectra for the measurements on 20 µM I3 (3.6 nmol per sample) look similar to the latter
described; again for lower pH lower than 6.0 the extremes can be observed at 288 and 257 nm and
for pH higher than 6.0 at 273 and 247 nm. Due to the higher concentration the noise is reduced
clearly. Many of the curves have two intersection points at 247 and 277.5 nm, similar to the ones
described above for the 6.3 µM measurement series. But for certain pH values the curve seems to be
shifted a little: for the first measurement series on 20 µM I3 the curve of pH 7.0 is a little too
protruding to cross the other curves at 247 and 277.5 nm. For the second measurement series the
intersection point at 247 nm remains unchanged, but the curves for pH 6.1 and 6.2 are shifted to
higher wavelengths and do not cross the other curves at 277.5 nm. Since the pH values which behave
differently are not the same for the two measurements and the curves for the 6.3 µM measurements
are not definitely interpretable because of the noise (so there is de facto no evidence whether the
isobestic point is as accurately defined as it appears) this is not taken as a hint for a third species
present but for some measurement mistake.
The pH values where the intensity at 288 nm is the highest, that means, the pH at which the i-motif
structure seems to be the most stable, differs slightly between the measurements. As it can be seen
in Fig. 9 and Fig. 11, two times the lowest pH that was measured seems to be the one with the most
stabilizing effect. For the third measurement it happens to be pH 5.5, whereas the pHs in the
proximity (pH 5.1, 5.25 and 5.6) are slightly lower in intensity. For the last measurement series pH 5.5
seems to be the second stabilizing (after pH 5.1). There is no clear explanation for this possible at the
moment; since CD spectroscopy is a very sensitive technique and the conversion from ellipticity to
17
— Results and discussion —
molar ellipticity increases the error from concentration differences enormously (a difference from
1 µM in concentration causes a difference of 100.000 deg·cm²/dmol in molar ellipticity) the actual
value of the molar ellipticity seems to be negligible. The change in ellipticities due to the increased
pH can be observed nevertheless.
pH-response
To monitor the pH response of the unfolding of the I3 sequence, the molar ellipticities at 288 nm
were plotted versus pH for all the three I3 measurement series (Fig. 12). It is expected that the
folding occurs cooperatively, i.e. a sigmoidal course can be seen in the data.
Fig. 12: Plot of the molar ellipticity *deg∙cm²/dmol+ at 288 nm against pH for I3. Plots for all
measurements (one for a concentration of 6.3 µM, two for 20 µM) are summarized here for better
comparability. For each measurement series a individual sigmoidal fitting with OriginPro 9G was
executed. The midpoint of the folding transition is labeled by a vertical line and R² (coefficient of
determination) for each plot is given.
The sigmoidal fit was performed with the software OriginPro 9G (provided by OriginLab,
Northampton) for each data set. As fitting model the “DoseResp” model (used for describing
apparent two-state models) was used that aligns the data to a function with the equation
𝑦 = 𝐴1 +
𝐴2−𝐴1
1+10 log 𝑥0−𝑥 𝑝
(equation 5)
A1 refers to the bottom asymptote, A2 to the top asymptote, the center (i.e. the inflection point) is
specified by log x0 and p is the so-called hill slope. The bottom and top asymptote are also called
“plateau” and are defined respectively by 100% folded and 100% unfolded structure. The steep
region between the plateaus represents the part where the folding occurs, i.e. the pH response
range. This part can be described quantitatively by the hill slope p; the higher it is, the steeper is the
part of the curve that is correlated to the folding in i-motif structure, or in other words, the higher
18
— Results and discussion —
the absolute value of p, the higher the cooperativity of the folding processes. log x0 is the midpoint
of the transition.
The fitting parameters for the three individual fits for the I3 sequence are summarized in Tab. 3.
Tab. 3: Fitting parameters for the I3 pH response curves.
sequence
A1
A2
p
log x0
R²
I3 (6.3 µM)
104803 ± 11626
510315 ± 12856
-3.361 ± 0.592
5.938 ± 0.026
0.987
I3 (20 µM. 1)
241217 ± 18971
932722 ± 20592
-3.358 ± 0.002
6.003 ± 0.024
0.989
I3 (20µM. 2)
227255 ± 113642
12099550 ± 58533
- 3.190 ± 1.186
5.931 ± 0.058
0.924
The three fitting curves show a sigmoidal course, which means that the folding is a cooperative
process. This is also indicated by the value >1 for the hill slope p. The look of the three curves is
differing to a great extent: although the ellipticity values have been converted into molar ellipticities
and the concentration differences should be excluded, the values for the molar ellipticity diverge
much. The values for the hill slope and for the inflection point, however, are only slightly varying,
which means that the interesting part of the plot, the part where the i-motif is unfolding, is still
reliable. Since there is no other difference between the samples (i.e. the experimental conditions –
temperature, buffer concentration – are maintained), some diversity in the concentrations must be
the reason for the different molar ellipticities (as described above, little errors cause contribute
largely to the molar ellipticity values); that the part of the plot corresponding to the folding process is
so equal in all cases could be a hint that the unfolding is concentration-independent. Independency
in concentrations is normally a hint for an intramolecular folding process which has been observed
for I3 several times (Manzini et al. 1994) (Zhou et al. 2010) (Mergny et al. 1995).
Taking all the data together, the pH range where the folding of the I3 sequence occurs can be
localized between pH 5.8 and pH 6.2. The midpoint of the transition can be settled at 6.0. These
results are in agreement with literature (Li et al. 2002) (Mergny et al. 1995) and will be taken as
reference for the following considerations on the other sequences.
19
— Results and discussion —
4.1.1.
I4 and I6: effect of the elongation of the cytosine-rich tracts
I4
Recently the work by Kwok et al. (Kwok et al. 2013) showed that the response range of G-quadruplex
RNA biosensors to K+ ions can be broadened by reducing the cooperativity of the RNA folding by
introducing negative cooperativity. Extending the g-rich tract leads to folding of several
intermediates which is responsible for a broadening of the response range about several magnitudes
in K+titration experiments. By elongating each c-rich tract in the I3 structure with one cytosine for I4
and with three cytosines for I6 we wanted to check whether the same idea can be transposed to crich DNA, because broadening the pH-response range would be of interest for pH-induced i-motif
biosensors.
I4 CD spectra
First CD spectra were recorded to check whether i-motif formation occurs in the I4 sequence. The CD
spectra obtained from the measurements at various pH values between 5.1 and 7.0 are shown in
Fig. 13.
Fig. 13: CD spectra of the measurement series on I4 (with 2.4 nmol DNA per sample). The ellipticity [mdeg]
in a wavelength range of 220 to 330 nm at different pH (pH 5.1 – 7.0) was measured and converted into
molar ellipticity *deg∙cm²/dmol+.The wavelengths that indicate the presence of i-motif are marked by blue
lines, the wavelengths that indicate the unfolded structure by green ones. The isosbestic points are either
marked by one of these or by a dashed line.
The ellipticity bands at 288 nm and 257 nm indicate that i-motif formation occurs at pH values lower
than 6.4. The structure seems to be most stabilized for pH between 5.1 and 5.5, whereas the
ellipticity values slowly decrease for pHs between 5.5 and 6.4. For pHs higher than 6.5, there seem to
be no folded i-motif anymore as there are only strong ellipticity bands at 273 and 247 nm.
The curves have two isosbestic points at about 277.5 and 241 nm, which indicates, as for I3, that
there are only two populations present, namely folded and not folded. The second one seems to be
20
— Results and discussion —
shifted a little to lower wavelengths compared with the isosbestic points which can be seen in the I3
CD spectra, but the ones for I3 are not so clearly defined as the ones for I4, so they may be the same
as well.
I4 pH-response
As for I3, the molar ellipticity at 288 nm (as this is the wavelength that is an indicator for i-motif
formation) in dependency of the pH value was plotted to investigate the pH-response of the DNA
(Fig. 14).
Fig. 14: Plot of the molar ellipticity [deg cm²/dmol] at 288 nm against pH for I4. The dark blue curve shows
the “DoseResp” fitting that was attached to the data, the light blue one shows a “BiDoseResp” fitting, i.e.
a fitting for a three-state model instead of a two-state model as for the “DoseResp”. The midpoints of the
transition are marked by horizontal lines that are labeled with the corresponding pH values. Also the R²
value is given for both fittings.
As can be seen in Fig. 14, two different fitting models were applied to the data. The first one
(represented by the dark blue line) is the basic “DoseResp” fitting model that was already used for
the fitting of the I3 data and which is used for describing two-state models. The second one
(“BiDoseResp”) is corresponding to an apparent three-state model, i.e. a model for an unfolding via
an intermediate. The data fits much better to the three-state model than to the two-state model
(which can be derived from the values of R², which is about 0.99 for the three-state model, but only
0.95 for the other one). Surprisingly, Kwok and coworkers observed a three-state model for a
sequence with four guanosines in the g-quadruplex forming structure. There the g-tetrad formed an
alternative structure (g-rich sequences can form many different g-quadruplex structures, e.g. with
different loop topologies, or parallel and antiparallel quadruplexes (Burge et al. 2006)), before it
unfolded completely. Because of the existence of the intermediate step, the folding range (due to an
increase in the concentration of K+, which is necessary for the g-tetrad folding) broadened (Kwok et
al. 2013). But for the i-motif there is no such intermediate known at the moment (at least not for
21
— Results and discussion —
intramolecularly foldings; Kanaori et al. found evidence for at least three different possible i-motif
structures built intermolecularly by four single-stranded c-rich tracts (Kanaori et al. 2001)), so there is
no explanation at the moment why the folding should follow a three-state model. Because of that,
new samples of I4 for pH 5.6 to 6.0 were prepared and measured. The fittings derived from the
combination of the old values with the ones newly measured are shown in Fig. 15.
Fig. 15: Plot of the molar ellipticity [deg cm²/dmol] at 288 nm against pH for I4 with new values for pH 5.6
to 6.0. The dark blue curve shows the “DoseResp” fitting that was attached to the data, the light blue one
shows a “BiDoseResp” fitting, i.e. a fitting for a three-state model instead of a two-state model as for the
“DoseResp”. The midpoints of the transition are marked by horizontal lines that are labeled with the
corresponding pH values. Also the R² value is given for both fittings. The red dot is from the value
obtained for pH 6,2 that is outstanding for both fitting models.
The R² values for the both fittings (R² = 0.94356 for the two-state model and R² = 0.9975 for the
three-state model) show that with the ellipticity values obtained from the newly prepared samples
the three-state model seems to be even more convincing than before. The plateau region that would
belong to the intermediate folding population (pH 5.6 to 6.0) is even better defined than for the first
fitting in Fig. 15. So both fitting models will be taken into consideration to describe the pH-response
of I4 and to compare it with the results for I3 in the following section.
The “BiDoseResp” model of OriginPro 9G is defined by equation 6:
𝑝
𝑦 = 𝐴1 + 𝐴2 − 𝐴1 [1+10 (log 𝑥01−𝑥)ℎ 1 +
1−𝑝
]
1+10 𝑙𝑜𝑔𝑥 02−𝑥 ℎ 2
(equation 6)
A1 and A2 are the bottom and top asymptotes (so they define the both plateaus), log x01 and log x02
are the two transition points, h1 and h2 are the hill slopes for the both steep regions, and p is a
proportion factor. The fitting parameter for both fits on both data sets are given in Tab. 4 and
Tab. 5.
22
— Results and discussion —
Tab. 4: Fitting parameters for the two-state model fitting for both I4 data sets.
data set
A1
A2
p
log x0
R²
1
149423 ± 116214
1562020 ± 61012
-2.239 ± 0.554
6.335 ± 0.051
0.960
2
-50923 ± 353479
1738010 ± 206644
-1.088 ± 0.481
6.248 ± 0.156
0.944
Tab. 5: Fitting parameters for the three-state model fitting for both I4 data sets.
Data
A1
A2
log x01
log x02
h1
h2
p
R²
1
255341
± 34200
1651540
± 42759
5.594 ±
0.052
6.407 ±
0.019
-5.241 ±
3.506
-4.927 ±
0.848
0.277 ±
0.047
0.991
2
257098
± 17813
1641770
± 19293
5.557 ±
0.019
6.429 ±
0.010
-12.003
± 2.929
-5.315 ±
0.564
0.355 ±
0.564
0.998
set
Regarding the two-state model, both fittings show a reduced cooperativity of p = -2.239 ± 0.554 and
p = -1.088 ± 0.156, compared to I3- where p is between -3.190 ± 1.186 and -3.361 ± 0.592 (average
value -3.303 ± 0.593). The average value for both I4 data sets is -1.644 ± 1.035 (the error is so high,
because the fitting is not accurate) which suggests a reduction of cooperativity about roughly two
times compared to I3. The pH range is shifted to higher pH values with a transition point of about 6.3
and a pH response range from 6.0 to 6.8 for the fit seen in Fig. 14 or even from pH 5.5 to 6.9 for the
fit in Fig. 15.
For the three-state model, there are two steep regions with hill coefficients of -5.241 ± 3.506 and
-12.003 ± 2.929 for the lower one (between the plateau of the proposed folding intermediate and
the plateau of the unfolded population) and -4.972 ± 0.848 and -5.315 ± 0.564 for the upper one
(between the plateau of the folded population and the plateau of the folding intermediate). Both on
their own do show a higher cooperativity, although together the pH range is broadened to 5.4 to 6.6
(Fig. 14 and Fig. 15). But with the plateau in the middle there is a (as Kwok et al. call it) “dead zone”
(Kwok et al. 2013) existing, where a biosensor based on the I4 sequence would show no pH-response
at all (proposed that the three-state model would be correct).
Consequently experiments shall be performed again to confirm either the two-state or the threestate model; all the samples were remeasured once and showed the very same behavior as
previously, so measurement errors may be to exclude, but not errors by concentration differences. It
is conceivable that the cytidines in the sequence show more than one apparent pKa; consequently
hydrogen bondings between them were formed at different pH. Maybe for higher pH values some of
the cytidines are engaged in the loops which may cause stabilization that is displaced by hydrogen
bondings between hemiprotonated base pairs when the pH is increased further. Since the CD spectra
23
— Results and discussion —
showed only two isobestic points (which is a hint for a sample with only two populations) it is
expected nevertheless that the two-state model will be confirmed with a pH range that is broadened
and shifted to higher pHs.
I6
With I6, another sequence was analyzed that had an increased number of cytosines compared to the
I3 wild type sequence. The deliberations that led to this choice of sequence were the same as for I4.
Here, again, should be investigated whether elongation of the c-rich tract is able to broaden the pH
range of a possible i-motif based pH biosensor.
I6 CD spectra
CD spectroscopic measurements were performed to test the ability of the I6 sequence for i-motif
formation. The spectra for a measurement series with a DNA concentration of 10.64 µM per sample
are shown in Fig. 16.
Fig. 16: CD spectra of the measurement series on I6 (with 1.9 nmol DNA per sample). The ellipticity [mdeg]
in a wavelength range of 220 to 330 nm at different pH (5.1 – 7.0) was measured and converted into
molar ellipticity *deg∙cm²/dmol+. The wavelengths that indicate the presence of i-motif are marked by
blue lines, the wavelengths that indicate the unfolded structure by green ones. The isosbestic points are
either marked by one of these or by a dashed line.
The spectra contain the i-motif characteristic ellipticity bands at 288 and 263 nm which prove that I6
does form the i-motif. Noteworthy, the negative band as actually shifted towards higher wavelengths
about 6 nm compared to the spectra for I3 and I4. For pH values lower than 6.8 at least a little
fraction folded i-motif is present. But the reduction of the molar ellipticity seems not to be directly
connected to the reduction of the pH. For example, the spectrum of pH 6.4 is clear outstanding
between the curves of pH 5.25 and pH 5.5, whereas the spectrum at pH 5.35 is located between the
ones for pH 5.6 and 5.8. As this disarrangement of pH values can be easier seen from the plotting of
the molar ellipticities at 288 nm against pH, it will be discussed in this section (I6 pH-response).
24
— Results and discussion —
Except for the curves corresponding to pHs 6.8 and 7.0, all spectra have two points were they cross
each other: at 273 and 237 nm. Once again this can be taken as a hint for the formation of only one
folding product. Compared to the isosbestic points of I3 and I4, the one at the lower wavelength is
shifted about 5 to 10 nm to lower wavelengths. According to Berlett et al., there are two reasons
possible for a shift of the isosbestic wavelength: either the molar absorptivity of the precursor (the
folded structure) changes, or the fraction of the precursor that is converted to multiple products
changes (Berlett et al. 2000). Brahms et al. observed a wavelength shift of the isosbestic point to
lower wavelength during their CD studies on polyA oligonucleotides when they increased the chain
length; together with the isosbestic point, the wavelengths of the minima and maxima shifted slightly
to lower wavelength, and the positive ellipticity band rose in intensity, in fact more rapidly than the
negative band (Brahms et al. 1966). Comparing the spectra of I3, I4 and I6, a similar behavior can be
detected: for I3, the molar ellipticities range from -7.5·105 deg·cm²/dmol to 1.25·106 deg·cm²/dmol
(at least for one measurement series; for the other two the amplitude is lower), for I4 values
between -7.5·105 deg·cm²/dmol to 1.75·106 deg·cm²/dmol were measured and for I6 from
-2.5∙106 deg∙cm²/dmol to 6∙106 deg∙cm²/dmol. Brahms et al. attribute their observations to the
“nearest neighbor” theory of Tinoco et al. (Tinoco et al. 1963), who state that every absorption band
in the spectrum of the monomer will be splitted into N bands in the spectrum of the N-mer. The final
spectrum is formed by adding up all these N absorption bands, so it is only reasonable that the longer
sequences show the higher molar ellipticity values in the experiments.
25
— Results and discussion —
I6 pH-response
To analyze the unfolding of the i-motif due to an decrease of the H+-concentration, the molar
ellipticities at 288 nm received from the CD spectra were plotted against pH (Fig. 17)
Fig. 17: Plot of the molar ellipticity *deg∙cm²/dmol+ at 288 nm against pH for I6 (1.9 nmol DNA per
sample).
Unlike the equivalent plots for I3 and I4, no sigmoidal course can be detected. There are two
outstanding points for the lower pH region, namely the second and third pHs (5.15 and 5.25) for
which molar ellipticities almost as twice as high as for the surrounding pHs were measured; the same
for pH 6.5 and 6.7 with molar ellipticities around 5∙106 deg∙cm²/dmol and 3∙103 deg∙cm²/dmol,
whereas for the other pHs in the proximity only molar ellipticities of about 1∙106 deg∙cm²/dmol to
1.5∙106 deg∙cm²/dmol were observed. For the outstanding pHs, new samples were prepared which
showed roughly the same behavior (data not shown). If pH 5.35 is taken as an measurement mistake
and pH 5.0 as a pH value that is so low that it is causing unfolding (which can occur if both cytosines
that may form a C∙C+ base pair are protonated, so the formation of hydrogen bond is no longer
possible), then still pH 6.5 and 6.7 cannot be explained. If all the outstanding values are excluded,
one may interpret the points that are left as belonging to a very broad transition part of a sigmoidal
curve with plateaus at pH values that were not measured here.
26
— Results and discussion —
The measurements on I6 were repeated, but with a lower sample concentration of 1.9 µM due to a
lack of DNA stock solution. The plot showing the pH response for these measurements is given in
Fig. 18.
Fig. 18: Plot of the molar ellipticity *deg∙cm²/dmol+ at 288 nm against pH for I6 (0.3 nmol DNA per
sample).
Again no sigmoidal course can be identified, although this time only one point is clearly outstanding
(pH 5.6). The magnitudes of the molar ellipticities are similar to the ones from the first
measurements, but a comparison of both (Fig. 19) reveals no other similarity.
Fig. 19: Comparison of both pH response curves (molar ellipticity vs. pH) for both I6 measurement series.
The CD spectra can only give a hint whether i-motif folding occurs or not. Since it was not possible to
prepare new samples in sufficient concentration to check the relevance of the outstanding points,
27
— Results and discussion —
the measurements should be re-performed. In addition, the new study should include more acidic pH
values to check whether a plateau region can be reached for pH values lower than 5.0.
28
— Results and discussion —
4.1.2. I3Me2 and I3Me4: effect of methylation on the 5-position of cytidine
Various studies on c-rich structures containing methylated cytidines have been reported - most of
them concern DNA triplex formations. A triplex is formed when a single strand that contains either
only pyrimidines or only purines binds via Hoogsteen base pairs to a duplex that is made of
homopurine and homopyrimidine strands as well (Povsic und Dervan 1989) (see section 2.2.1). For
such GC-triplexes it was shown that methylations stabilize triplex-DNA under acid conditions; in one
case the pH response range of the triplex was broadened by one pH unit and the melting
temperature was enhanced strongly about 10°C (Xodo et al. 1991). It was thus considered that
methylation could stabilize the i-motif structure as well. Besides, the introduction of a methyl group
was expected to alter the pKa of the cytidine and therefore has influence on the hydrogen bonding
between the hemiprotonated cytidines. In fact, the literature provides different statements on the
pKa of methylated cytidine: Moser et al. state a pKa shift of +0,7 (Moser et al. 2009) (the pKa of
unmodified cytidine is here stated as 4,8), whereas Mergny et al. observed no pKa shift at all when
they replaced all cytidines by methylated cytidines in a c-rich sequence that was NMR proofed to
form the i-motif(Mergny et al. 1995). In general, a methyl group has an electron-donating effect on
aromatic rings and is therefore expected to enhance the pKa value. An enhanced pKa means that the
hemiprotonated cytidines are stabilized towards more basic conditions, hence we can assume that
the pH-response range should be shifted towards higher pH values.
Two different oligonucleotides that contained 5-methylcytosine were analyzed: I3Me2 with two
substituted cytidines that are involved in the same base pair, I3Me4 with four 5-methylcytosines,
engaged in two hydrogen bonds. The5- methylated cytidines were placed in the middle of the CCCtrack to reduce interference with the adjacent TAA-loops which may have a destabilizing effect.
29
— Results and discussion —
I3Me2 and I3Me4 CD spectra
CD spectra at pHs between 5.1 and 7.0 were recorded to provide evidence for i-motif formation and
monitor the pH-induced unfolding. The resulting spectra are shown in Fig. 20 and Fig. 21.
Fig. 20: CD spectra of I3Me2 (with 2.8 nmol DNA per sample). The ellipticity [mdeg] in a wavelength range
of 220 to 330 nm at different pH (4.8 - 6.8) was measured and converted into molar ellipticity
*deg∙cm²/dmol+. The wavelengths that indicate the presence of i-motif are marked by blue lines, the
wavelengths that indicate the unfolded structure by green ones. The isosbestic points are either marked
by one of these or by a dashed line.
Fig. 21: CD spectra of I3Me4 (with 3.5 nmol DNA per sample). The ellipticity [mdeg] in a wavelength range
of 220 to 330 nm at different pH (5.1 - 7.0) was measured and converted into molar ellipticity [deg
cm²/dmol]. The wavelengths that indicate the presence of i-motif are marked by blue lines, the
wavelengths that indicate the unfolded structure by green ones. The isosbestic points are either marked
by one of these or by a dashed line.
Both spectra show the ellipticity bands in the wavelength regions that were described previously as
corresponding to i-motif folding: a positive band at roughly about 288 nm, a negative band between
255 and 260 nm. The shift to lower wavelengths (the unfolded sequence has its maximum at 273 nm
30
— Results and discussion —
and its minimum at 247 nm), indicating the unfolding of the structure, begins for I3Me2 at pH values
higher than 6.0, for I3Me4 at pHs higher than 6.1. For both sequences the maxima and minima of the
ellipticity bands seem to be shifted slightly to higher wavelengths (compared to the previously
described observations for I3, I4 and I6; except for the 257 nm band that stays the same or has a little
shift to the left), which may be caused by some change in absorpticity due to the introduced
methylations. For both sequences isosbestic points are observable, which is a hint for the presence of
only two species in the sample, i. e. i-motif and unfolded DNA. For I3Me2, the wavelengths of the
isosbestic points (278 nm and 247 nm) are similar to the ones observed for I3, whereas for I3Me4 the
wavelength of the isosbestic point in the lower wavelength range is shifted to lower wavelength (in
the order of 7 nm to 240 nm). Similar results were observed for I6, and as described there the reason
may be an altered molar absorptivity of the precursor. Interestingly, Mergny et al. observed a shift of
the isosbestic point to longer wavelengths when comparing the spectra of a c-rich structure with that
of its derivative, where all cytidines were replaced by methylcytidines (Mergny et al. 1995). So maybe
this wavelength shift is sequence-dependent.
The range of the molar absorptivity is different for both sequences: for I3Me2 the observed
minimum at pH 5.1 is about -7.5∙105 deg∙cm²/dmol, the maximum at 1.25∙106 deg∙cm²/dmol and is
therefore in the same scope as was observed for I3. The ellipticity range for I3Me4 in contrast is
located between -4∙105 deg∙cm²/dmol and 7∙105 deg∙cm²/dmol, so only half as much. Since the
number of nucleotides is not changed from I3 to I3Me2 or I3Me4, the nearest neighbor theory of
Tinoco et al. (Tinoco et al. 1963) that was used to describe the differences for the I3, I4 and I6 molar
ellipticity range (see section 4.1.2)cannot be used as an explanation here.
There is another concept that is sometimes used to describe CD spectra (or spectra obtained from
rotatory dispersion spectroscopy): the concept of rotational strength, which creates a quantitative
magnitude that describes the energy of the electronic transition from the ground state to the excited
state (depending on both the electric and magnet dipole moment). It takes not only the maximum
ellipticity into account, but also the width of the ellipticity band, as it is expressed by the following
equation (if the band has a Gaussian shape, which is the case, at least for the bands at 288 nm)
(Beychok 1966):
𝑅𝑘 ≈ 0.696 ∙ 10−43 ∙
𝜋 ∙ 𝜃𝑘0 ∙
∆0𝑘
𝜆 0𝑘
(equation 7)
Rk is the rotational strength, 𝜃𝑘0 is the value of the ellipticity at the wavelength of the band
maximum, 𝜆0𝑘 is the wavelength of maximum ellipticity and ∆0𝑘 is the band width (the wavelength
interval between 𝜆0𝑘 and the wavelength at which the ellipticity is e-1 times 𝜃𝑘0 ). It is derived from
the more general equation 8,
31
— Results and discussion —
𝑅𝑘 =
3ℎ𝑐
8𝜋𝑁
[θ]𝑘
𝑑𝜆
𝜆
(equation 8)
where h is Planck’s constant, c the velocity of light, N the number of absorbing molecules per cubic
centimeter and [𝜃]𝑘 the ellipticity.
Calculating the rotational strengths of each molecule lies far beyond the scope of this work; the
intention was to show that the CD signal is not only describable by the bare ellipticity value itself, but
also has the width of the ellipticity band and the wavelength where it occurs to be taken into
consideration. Equation 7 shows that the rotational strength is the larger the maximum ellipticity and
the band width are, that means, the broader the ellipticity band is. The rotational strength also
increases with decreasing wavelength corresponding to the maximum ellipticity. It seems therefore
possible that a decrease in maximum ellipticity is somewhat compensated by a cantilevered band
shape as well as by a shift of the wavelength were the maximum ellipticity is observed. Nevertheless
due to intrinsic molecule properties, the rotational strength of I6 seems to be thoroughgoing high
compared to the ones of the other sequences.
32
— Results and discussion —
I3Me2 and I3Me4 pH-response
To monitor the pH-dependency of the unfolding of I3Me2 and I3Me4, the molar ellipticities at
288 nm (the wavelength where the maximum ellipticity is obtained if i-motif is present) were plotted
against pH. For better comparability, the curves of I3, I3Me2 and I3Me4 were plotted together
(Fig. 22).
Fig. 22: Plot of the molar ellipticity *deg∙cm²/dmol+ at 288 nm against pH for I3, I3Me2 and I3Me4. The
transition points are marked by vertical lines with corresponding pH values. Two-state “DoseResp” models
for all sequences were separately applied to the data; the corresponding R² are also given.
For both I3Me2 and I3Me4 a sigmoidal course of the pH-induced unfolding is obtained. This means
that the unfolding is a cooperative process that only includes a folded and an unfolded state (twostate model). Sigmoidal fits were attached to the data by the use of the software OriginPro 9G
(provided by OriginLab, Northampton). The fitting parameters (their relevance is described in the
sections before) are given in Tab. 6.
Tab. 6: Fitting parameter for the pH response curves of the CD measurements on I3Me2 and I3Me4.
Sequence A1
A2
p
log x0
R²
I3Me2
298846 ± 22467
1218350 ± 17828
-3.102 ± 0.357
6.087 ± 0.019
0.992
I3Me4
204727 ± 8139
642651 ± 6596
-3.466 ± 0.315
6.156 ± 0.012
0.995
The R² values show that the fit data is quite close to the measured data. Compared to I3, the
cooperativity of I3Me2 is reduced with a hill slope of -3.102 ± 0.357 (average value for I3:
-3.303 ± 0.593), the one of I3Me4 is with p = -3.466 ± 0.315 enhanced. This corresponds to the
broader pH range for I3Me2 (roughly from pH 5.8 to 6.3) and the tighter one for I3Me4 (from 6.0 to
6.4). Additionally, the pH of transition is shifted slightly to higher pH values. Both effects are not
strong, which seems to be in accord with the findings of Mergny et al., who observed no significant
33
— Results and discussion —
influence of methylations on another i-motif forming sequence (Mergny et al. 1995). Still, the
broadening of the pH range of I3Me2 could be of use in order to find possible structures to utilize as
pH induced biosensors, if an i-motif forming sequence was found with a higher pKa. Besides, another
effect may be achieved if the positions of the methylcytidines in the I3Me4 structure were altered,
since the methylations in the structure as it was used are located on cytosines that are spatial close
to each other, which may have induced a steric hindrance. This can be checked with a modeling
software quite easily. It may be possible that with methylations at other positions a more convincing
effect is achievable.
34
— Results and discussion —
4.1.3.
I3Br2 and I3Br4: effect of bromine substitution in the 5-position of cytidine
Besides I3Me2 and I3Me4, sequences that contain methylated cytidines, the effect of a bromine
substitution on the 5-nitrogen atom of cytidine was investigated. The same position were chosen
were substitution as previously with the 5-MeC for the same reasons (to avoid interference with the
TAA-loops). Bromine substitutions are not common in the eucaryotic DNA; bromination of thymidine
indeed leads to 5-bromouracil, which can cause mutagenesis and is therefore possibly dangerous to
living organisms. In fact, bromination is able to stabilize poly(dG-dC) in the Z-DNA form, a left-handed
helical form of DNA with 12 base pairs per helical turn (Möller et al. 1984) (Ross et al. 1989). Native
calf thymus DNA was found as Z-DNA as it was brominated (Hasan und Ali 1990). Therefore an effect
of bromination on the i-motif folding is imaginable, especially because of it pKa lowering effect on
cytidine (pKa = 2.45) (Kulikowski und Shugar 1979).
There are at least two possible effects of the bromination to consider: first, bromine substitution
could have an influence on the hydrogen bonding between the hemiprotonated C-C base pair, as it
increases the acidity of the cytidine (Moser et al. state a pKa shift of -2.6 for 5-bromocytosine
compared with unsubstituted cytosine (Moser et al. 2009)). Second, it may stabilize the
hemiprotonated base pair in a manner shown in Fig. 23, by interaction with the adjacent amino
group attached to C4 of cytosine.
Fig. 23: Schematic representation of a hemiprotonated C∙C+ base pair in DNA i-motifs. In blue are the
three hydrogen bondings between the two cytidines, in green a potential hydrogen bonding between the
bromine at C5 and the amino group at C4.
35
— Results and discussion —
I3Br2 and I3Br4 CD spectra
CD spectra in the wavelength range from 220 to 330 nm were recorded to test the ability of I3Br2
and I3Br4 to form the i-motif. The resulting spectra are shown in Fig. 24 and Fig. 25.
Fig. 24: CD spectra of I3Br2 (with 1.1 nmol DNA per sample). The ellipticity [mdeg] in a wavelength range
of 220 to 330 nm at different pH (5.1 - 7.0) was measured and converted into molar ellipticity
*deg∙cm²/dmol+. The wavelengths that indicate the presence of i-motif are marked by blue lines, the
wavelengths that indicate the unfolded structure by green ones. The isosbestic points are either marked
by one of these or by a dashed line.
Fig. 25: CD spectra of I3Br4 (with 4.3 nmol DNA per sample). The ellipticity [mdeg] in a wavelength range
of 220 to 330 nm at different pH (4.8 - 7.0) was measured and converted into molar ellipticity
*deg∙cm²/dmol]. The wavelengths that indicate the presence of i-motif are marked by blue lines, the
wavelengths that indicate the unfolded structure by green ones. The isosbestic points are either marked
by one of these or by a dashed line.
The spectra look similar to the ones for the other sequences: ellipticity bands at 288 nm and 257 nm
are present, so i-motif folding does occur for I3Br2 and I3Br4. For I3Br2, it seems to be stable for pH
values below 5.6, whereas the bands at 273 and 247 nm demonstrate that the unfolding is already
36
— Results and discussion —
completed for pHs above 6.0. The unfolding thus takes place very rapidly. A similar behavior can be
observed for I3Br4: here the unfolding seems to be finished above pH 5.8. Concerning the locations
of the ellipticity bands, similarities to the spectra of I3Me2 and I3Me4 can be detected: except for
the band at 258 nm which is shifted slightly to shorter wavelengths, the maxima of the ellipticity
appear at slightly higher wavelengths. As explained for I4 and I6, this may be due to a change in
absorptivity of the molecules. Isosbestic points can be observed for I3Br2 and I3Br4 as well, for the
first at 277 nm and 241 nm, for the latter at 280 nm and 239 nm. For the ones at lower wavelengths,
the same tendency as for I3Me2 and I3Me4 is detectable: a shift to lower wavelength, as well
compared to I3 as also among themselves. Introducing the substitution seems to alter the
absorptivity of the DNA, and the effect is enhanced with the number of substitutions. In contrast, the
other isosbestic point is, as far as I3Br4 is concerned, are shifted to higher wavelengths – indeed each
spectrum seems to be somewhat broadened. The reason for this may be that not the whole pHresponse range is monitored and maybe sharper bands would be obtained for measurements at a
lower pH.
The molar ellipticity range is the lowest compared to all the other structures that were analyzed,
from -1.5∙106 deg∙cm²/dmol to 2∙106 deg∙cm²/dmol for I3Br2 and from -3∙105 deg∙cm²/dmol to
4∙105 deg∙cm²/dmol for I3Br4. It is eye-catching that the absolute value for the minimum ellipticity is
close to the maximum ellipticity for these sequences, so the obtained spectra almost have a
symmetrical shape. For all the other sequences the maximum ellipticity was clearly higher than the
absolute value of the minimum ellipticity. The ratios of the extreme ellipticities of each sequence
compared to the ones observed for I3 and the ratio of maximum and minimum molar ellipticity
within each sequence are summarized in Tab. 7 for better visualization.
Tab. 7: Comparison of the maximum and minimum molar ellipticities of each sequence to that of I3 and ratio of
maximum to minimum molar ellipticity for each sequence. ”x” refers to each particular sequence.
sequence
Minimum
ellipticity
[deg ∙ cm²/dmol]
-7.5 ∙ 10
5
-7.5 ∙ 10
5
-2.5 ∙ 10
6
I3Me2
-7.5 ∙ 10
5
I3Me4
-4 ∙ 10
I3Br2
-1.25 ∙ 10
I3
I4
I6
I3Br4
-3 ∙ 10
Maximum
ellipticity
[deg ∙ cm²/dmol]
1:1
1.25 ∙ 10
1:1
3.33 : 1
5
5
Minimum
ellipticity
ratio
(x : 1)
6
Ratio max.
ellipticity/min.
ellipticity
(x : 1)
6
1:1
1.67 : 1
6
1.4 : 1
2.33 : 1
4.8 : 1
2.4 : 1
1:1
1.67 : 1
5
0.56 : 1
1.75 : 1
6
1.6 : 1
1.6 : 1
5
0.32 : 1
1.3 : 1
1.75 ∙ 10
6
6 ∙ 10
6
1:1
1.25 ∙ 10
0.53 : 1
7 ∙ 10
1.67 : 1
2 ∙ 10
0.4 : 1
Maximum
ellipticity
ratio
4 ∙ 10
37
— Results and discussion —
From the data summarized in Tab. 7, several observations can be made: first, for all sequences the
maximum molar ellipticity was higher than the absolute value of the minimum ellipticity. But
whereas for I3Me4 the ratio of both increases compared to I3Me2, it decreases in the step from
I3Br2 to I3Br4. Also the differences in the total values are much bigger in comparing I3Br2 and I3Br4
as in comparing I3Me2 and I3Me4. Together with the broadening of the spectral bands, this may lead
to the conclusion that the effect of the bromine substitution on the absorptivity of the sequence
might be different than the methylation effect. Since CD spectroscopy is particularly sensitive to
secondary structures (Sprecher et al. 1979), this may imply that here changes are happening, too.
I3Br2 and I3Br4 pH-response range
The pH dependency plots (plots of the molar ellipticity at 288 nm vs. pH) for I3, I3Br2 and I3Br4 are
shown in Fig. 26.
Fig. 26: Plot of the molar ellipticity *deg∙cm²/dmol+ at 288 nm against pH for I3, I3Br2 and I3Br4. The
transition points are marked by vertical lines with corresponding pH values. Two state “DoseResp” models
for all sequences were separately applied to the data; the corresponding R² are also given.
For both I3Br2 and I3Br4, it is possible to apply a sigmoidal fit to the data with satisfying
determination coefficients of R² = 0.998 for I3Br2 and R² = 0.992 for I3Br4. Again, as for all the other
sequences, the pH-induced unfolding of the i-motif seems to be cooperative. The fitting parameters
(obtained by fitting with OriginPro 9G(OriginLab, Northhampton)) of both fits are specified in Tab. 8.
Tab. 8: Fitting parameter for the pH response curves of the CD measurements on I3Br2 and I3Br4.
Sequence A1
A2
p
log x0
R²
I3Br2
571589 ± 15089
1991980 ± 18624
-3.800 ± 0.271
5.744 ± 0.011
0.998
I3Br4
163068 ± 3036
442728 ± 25579
-1.803 ± 0.231
5.134 ± 0.055
0.992
38
— Results and discussion —
Compared to the hill coefficient obtained for I3, which was about -3.303 ± 0,593 (average of three
fittings), the cooperativity of unfolding of I3Br2 seems to be higher (p = -3.800 ± 0.271), the
cooperativity for the I3Br4 unfolding instead is greatly reduced, indicated by a hill coefficient of
-1,.803 ± 0.055. The pH range of folding is therefore for I3Br2 narrower than for I3 and for I3Br4
broader, as it can be easily seen in Fig. 26. Indeed it may be possible that the transition region for
I3Br4 is even broader, because the measured pH values may have been too high to reach even the
beginning of the upper plateau region (so that the value at pH 4.8 still belongs to the transition
region). Going to lower pH was not possible because of a lack of DNA. The transition pHs are for both
sequences shifted to lower pH values. The potential broadening of the pH range for I3Br4 could be
valuable for nanodevices that are used to monitor pH regions below pH 5.4. It is interesting to notice
that the effect of the bromination due to the pH response of the i-motif is more important than the
effect of the methylations.
39
— Results and discussion —
4.1.4. Interim Summary
Tab. 9 summarizes the results obtained from the CD spectroscopic measurements so far.
Tab. 9: Summary of the results obtained from CD spectroscopy for all sequences.
Sequence
pH-response range
pH transition point
Cooperativity
compared to I3
I3
5,8 – 6,2
6,0
I4ª
5,4 – 6,6
6,3
reduced
I6
?
?
reduced
I3Me2
5,8 – 6,3
6,1
reduced
I3Me4
6,0 – 6,4
6,16
enhanced
I3Br2
5,6 – 6,0
5,7
enhanced
I3Br4
< 4,8 – 5,6
5,1
reduced
ªfor the two state model.
The diversification of the I3 sequence by elongation of the c-tracts and introducing of substitutions
had the initial ambition to broaden the pH response range of the i-motif, in order to produce a
possible application as a pH induced biosensor. The CD spectroscopy experiments showed that the
elongation of the tracts is able to both broaden the pH range and to shift the transition point to more
basic pH, i.e. to shift the pKa of the cytidines. The 5MeC ∙ 5MeC+ base pairs shift the pKa slightly to
higher values as well, but at the same time the pH range is narrowed. The 5BrC ∙ 5BrC+ base pairs
finally shift the pKa greatly to lower values. Hence both initial considerations about 5MeC and 5BrC
concerning the pKa of cytidine were correct.
40
— Results and discussion —
4.2.
Stoichiometry of the i-motif folding
The aim of the following experiments was to determine the number of DNA strands participating in
the formation of i-motif for each particular sequence. The i-motif structure has been demonstrated
for many oligonucleotides containing c-stretches of variable length. For the sequences d(T2C8T2),
d(TC5) and d(C4TC4) i-motifs were proven that contained four single-stranded DNA molecules (Leroy
et al. 1993), whereas many DNA strands containing four tracts of cytosine form intramolecular imotifs, for example GC(TC3)3TCCT(TC3)3C (Manzini et al. 1994), (C4G3]3C4TA and C2T3C2T4C2T3C2 (Zhou
et al. 2010). DNA strands with two stretches of cytidines with an intermediate linker can in many
cases be observed as dimeric i-motifs (for example 5mCCT3AC2) (Guéron und Leroy 2000). Depending
on conditions, some c-rich structures show more than one structure: for d(C2T2C2T4C2T2C2), 60%
eluted as dimer, 40% as monomer in gel filtration chromatography (Mergny et al. 1995). For the
human telomeric c-rich sequence, d(C3TA2)3C3 (I3), it was shown various times that the i-motif folding
occurs intramolecularly (Manzini et al. 1994) (Mergny et al. 1995) (Zhou et al. 2010). For the
approach to find a sequence that can be used as pH-sensitive nanodevice, it is of interest whether
the i-motif is formed by only one DNA strand, or if several strands together form the i-motif
structure. For easier handling, a nanodevice should be based on an monomolecular i-motif. Since it
has been shown that I3 at acidic conditions forms an intramolecular i-motif, it can be used as
reference in gel electrophoretic experiments.
41
— Results and discussion —
4.2.1.
Native gel electrophoresis under acidic conditions (pH 5.0)
The electrophoretic mobility of all sequences was compared to that of I3 on a native 20%
polyacrylamide gel. The pH of the gel was set to pH 5.0 to be sure that the conditions are favorable
for the i-motif formation. The picture of the gel under UV light is shown in Fig. 27.
Fig. 27: Native polyacrylamide gel (pH 5.0) for all sequences. The DNA was visualized under UV light.
A 2:1 mixture of I3 and its complementary strand (“G3”, AT2(G3AT2)3), which are known to form a
double helix consisting of GC-Watson-Crick base pairs, was used as a size marker, assigning the
horizontal positions of a 21-mer and a 42-mer strand. The dye front migrated homogeneous, so it is
possible to estimate the molecularity of the i-motifs formed by each sequence in comparison with
the bands obtained for the size marker.
In lane 1, the apparent DNA band corresponds to I3. It is exact at the same level as the band to the
left of it which is also due to I3. In lane 2 and 3, the bands of I4 and I6 show a migration delay which
is most likely caused by their larger size which influences their electrophoretic mobility. If the reason
for the delay was a bi- or higher molecularity, the shift would have been to be stronger since a bi-I4
would have behaved similarly to a 50-mer and bi-I6 to a 66-mer. Actually sequences containing four
C-tracts composed of four cytidines have already been reported to form intramolecular i-motifs
(Mergny und Lacroix 1998) (Zhou et al. 2010).
Regarding I3Me2 and I3Me4 (lanes 4 and 5), a migration delay could be observed as well, but also
with only a minor shift which is too little to likely be caused by a dimer formation. It is probably
caused by a slight difference in structure compared to I3 as a result of the insertion of 5MeC. The
migrations bands of the two methylated sequences are at the same level, so the effect of the twofold
methylation at the migration rate of I3 is much bigger than the twofold methylation of the already
twofold methylated I3Me2. Mergny et al. showed for both the sequences d(TC2TC2T4C2TC2T) and
d(T(5MeC)2T(5MeC)2T4(5MeC)2T(5MeC)2T) that intramolecular i-motifs were formed (Mergny et al.
42
— Results and discussion —
1995), as well as Manzini et al. for d(5MeCC5MeC)3T5MeCCT(5MeCC5MeC)3C (Manzini et al. 1994).
All together, it is concluded that the i-motif formed by I3Me2 and I3Me4 occurs intramolecularly.
I3Br2 and I3Br4 (lanes 6 and 7) show as well a slower migration than I3, but even slower than I3Me2
and I3Me4. However, when I3Br2 and I3Br4 are compared to one another, both DNAs present the
same mobility. The last observation was unexpected at first, because the CD spectra showed that
I3Br4 is mainly unfolded pH 5.0. But the CD spectra have been recorded at 25°C, and during the gel
electrophoresis water cooling was applied to the apparatus so the lower temperature may have
stabilized the i-motif. It has been shown several times that lower temperatures have this stabilizing
effect and are able to compensate pH values that are unfavorable to some extent (Manzini et al.
1994) (Zhou et al. 2010). So it seems reasonable that i-motif folding for I3Br2 and I3Br4 occurs also
intramolecularly. Above the bands referring to the intramolecular i-motif, for each sequence a lighter
band is observable that is roughly on the same level as the 42-mer (I3/G3). These may correspond to
a bimolecular i-motif. To get distinctness, another gel electrophoresis was performed, but this time
under basic conditions (pH 8.5).
43
— Results and discussion —
4.2.2. Native gel electrophoresis under basic conditions (pH 8.5)
As described above, another gel electrophoresis was executed, this time under basic conditions
(pH 8.5). Only I3 and I3Br2 were loaded due to a lack of I3Br4 (Fig. 28).
Fig. 28: Native polyacrylamide gel for I3 and I3Br2 under basic conditions (pH 8.5). The DNA was visualized
under UV light.
Under these basic conditions I3 is present as a coil structure. I3Br2 shows a band with the same
mobility as I3, so I3Br2 is unfolded as well. However, it presents a second band higher in the gel, just
below the dye front, exactly as in the folding-gel. It is unlikely that we get a long DNA impurity from
the synthesis, which would mean that we actually get a stranger structure stable at pH 8.5 and
furthermore at pH 5.0. This consideration will not be further commented and according to its
marginal presence we will consider that I3Br2 and I3Br4 sequences mainly show an intramolecular imotif organization. However this odd result should be further studied elsewhere.
44
— Results and discussion —
4.2.3. Melting curves of I3Br2 at different concentrations.
Since the stoichiometry interpretations obtained from the gel electrophoresis could seem awkward,
we decided to check it by another experiment.
It is possible to distinguish between an inter- and intramolecular folding by melting curve
measurements, because the melting temperature is concentration-independent in case of a
monomolecular folding mechanism (i.e. intramolecular), but it is concentration-dependent if the
folding is bi- or higher molecular as for a intermolecular i-motif formation (Mergny et al. 1995).
Therefore, to get evidence about the numbers of actors in the folding of I3Br2, melting curves with
different concentrations were measured. The results are shown in Fig. 29.
Fig. 29: Melting curves of I3Br2 with different concentrations (20 µM, 6,67 µM and 2 µM), all measured at
pH 5.4. The melting temperatures that were obtained by fitting with a two state model are marked with
vertical lines; also the corresponding R²are given.
The fitting was performed by a two-state fitting model as described previously. The melting curve for
a concentration of 2 µM shows high noise and therefore the fitting is not so accurate (R² = 0.959) as
for the other concentrations. The melting temperatures obtained from the fitting are not the same
(about 31.8°C for the 20 µM sample, 30.4°C for the 6.67 µM sample and 29.7°C for the 2 µM sample),
but including the errors it is not reasonable to speak of different melting points and therefore the
folding of I3Br2 is stated as intramolecularly.
45
— Results and discussion —
4.3.
CD Melting curves: thermal stability of the i-motifs
Unfolding of i-motif structures can, as for any secondary structure, be induced by an increase of
temperature. Monitoring a certain temperature-dependent property can reveal important
thermodynamic parameters of the molecule of interest concerning its thermal stability, for example
the Gibbs energy ΔG which defines for a possible reaction its favorableness. Therefore it can tell
whether a given system is stable under certain conditions or not. The Gibbs energy is defined by
equation 9 (all equations derived here are adapted from Mergny and Lacroix (2003), unless otherwise
stated):
∆𝐺 = −𝑅𝑇𝑙𝑛 𝐾
(equation 9)
R is here the perfect gas constant with a value of 8.314 J/(K∙mol), T is the temperature in K and K is
the equilibrium constant, a quantity that describes the composition of a system as the ratio of its
components:
𝐾=
[𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑠 ]
[𝑒𝑑𝑢𝑐𝑡𝑠 ]
(equation 10)
At this composition of the system where K equals 1, ΔG equals 0. At this state the reaction is at
equilibrium. Observing the unfolding of monomeric i-motifs, where there is only one educt (the
folded structure) and one product (the unfolded DNA strand) involved, this equilibrium is reached
when the concentration of folded molecules is the same as the concentration of unfolded molecules.
So for a two-state “all-or-none” system (Mergny et al. 1995) (where outside of the transition part the
molecule is stated as either 100 percent folded or 100 percent unfolded) this is achieved in the very
middle of the steep transition region. The corresponding temperature is called the melting
temperature Tm.
At T > Tm, the corresponding Gibbs energy will be positive, indicating that the unfolding reaction is
favored opposite to the folding reaction, therefore the molecule is unfolded- and vice versa. So the
value of ΔG can be treated as a quantity that describes the stability of the i-motif at a given
temperature. Two fundamental principals contribute to it: first the attempt of every system to reach
the state with the lowest possible total energy, second the attempt to reach the state with the
highest possible disorder. The first is usually described by ΔH, the enthalpy; the latter one by ΔS, the
entropy. They contribute to ΔG as equation 11 describes:
∆𝐺 = ∆𝐻 − 𝑇∆𝑆
(equation 11)
Hence negative values for ΔH and positive ones for ΔS are favorable for reactions, i.e. the enthalpy
has to be reduced to favor a reaction; the entropy has to be enhanced.
46
— Results and discussion —
In these experiments the ellipticity at 288 nm (the wavelength where the maximum ellipticity is
observed if i-motif is present) as a function of temperature is recorded. For each temperature, the
folded fraction α can be calculated when assuming the highest obtained value as corresponding to
100 percent folded structure and the lowest obtained value to 100 percent unfolded structure. The
folded fraction can be transformed into the equilibrium constant K via K = α/(1-α). Plotting ln(K)
against 1/T, the so-called “van’t Hoff plot” is generated, corresponding to the following equation:
ln 𝐾 = −
∆𝐻°
𝑅
∙
1
𝑇
+
∆𝑆°
𝑅
(equation 12)
Thus it is expected to achieve a linear plot with a slope of -ΔH°/R and a y-axis-intercept
corresponding to ΔS°/R. With ΔH° and ΔS° known, the Gibbs energy at any temperature can be
determined.
Here experiments were carried out for each sequence on the one hand at a pH of 5.1, on the other
hand at pH 5.4. It was considered interesting to compare the effect of pH on the melting behavior as
coherences have been reported (Manzini et al. 1994) (Mergny und Lacroix 1998).
47
— Results and discussion —
4.3.1.
I3
Determination of melting temperatures
First the results for I3 should be discussed as it is the structure of reference. Fig. 30 shows the
melting curves obtained from the measurements on I3 for pH 5.1 and pH 5.4.
Fig. 30: Melting curves of I3 at pH 5.1 and pH 5.4. The measured ellipticity values were converted into the
ratio of folded to unfolded DNA. Sigmoidal fits were attached to the data; the melting temperature, which
is defined as the temperature at the transition point of the curve, is marked by vertical lines. R²
parameters for the fits are given.
Both curves show a cooperative profile as it is expected. The transition points of the fits, which
provide the melting temperatures, are at 40.5°C ± 0.04 for the measurement at pH 5.4 and at
48.2°C ± 0.07 for pH 5.1, thus a difference of 7.7°C ± 0.11 is observed. The fits were performed with
the software OriginPro 9G as already described. The fitting parameters of both fits are given in Tab.
10.
Tab. 10: Fitting parameter for the two-state model fits for I3 at pH 5.1 and 5.4.
sequence
pH
A1
A2
logx0
p
R²
I3
5.1
0.022 ± 0.002
0.955 ± 0.002
48.075 ± 0.070
-0.084 ± 0.001
0.997
I3
5.4
0.026 ± 0.001
0.960 ± 0.001
40.494 ± 0.042
-0.106 ± 0.001
0.999
Except for the hill coefficient p, all fittings parameters are similar for both pH values. At pH 5.4, the
absolute value is a little higher for p as in the fitting for pH 5.1, which means that the folding seems
to be a little more cooperative. So besides the lower melting temperature at pH 5.4, the melting is
completed in a shorter pH range. The dependency of the melting temperature on pH is described in
the literature as linear, at least in pH ranges not close to the pKa of the cytosines. Leroy et al.
observed for the (C3TA2)3C3 sequence in the pH range from pH 5.5 to 7.5 a melting temperature shift
48
— Results and discussion —
of about 22°C per pH unit (1994). Herein, we obtained a temperature shift of about 25.6°C per pH
unit, which is slightly higher. The reason for this may be that both pH or at least pH 5.1 lie outside the
range for a linear dependency of the melting temperature on pH. Mergny et al. observed for the
same sequence a melting temperature of 39°C for pH 6.0 (1995), Leroy et al. one of 29°C for pH 6.4
(Leroy et al. 1994). Manzini et al. measured a melting temperature of 53°C at pH 5.1 for the almost
identical sequence (C3TA2)C3T (1994). The melting temperature is highest when the pH equals the
pKa, because there the structure is hemiprotonated and both deprotonation and protonation are
unfavorable. Therefore it is expected that the melting temperature should be higher in the range
between pKa and the part of linear pH dependency than it was if linear dependency was given. So
the here obtained melting temperatures for I3 at pH 5.1 and pH 5.4 seem to be reliable.
Determination of thermodynamic parameters
As described in the introduction of this section, the data from the thermal denaturation experiments
can be used to calculate ΔH°, ΔS° and ΔG. The melting temperature is useful to get a general
conclusion on whether a structure is folded at a certain temperature or not, but a conclusion to the
general stability of the structure cannot be made so easily. Therefore it is useful to calculate the
Gibbs energy ΔG for any temperature of interest, which can give information about the forces that
are responsible for the stability (Mergny und Lacroix 2003). For DNA, enthalpic effects mostly come
from the stacking interactions between bases due to dipole interactions between the aromatic rings
(Yakovchuk et al. 2006), and not, as previously assumed, from the hydrogen bondings between the
bases (Watson und Crick 1953).Because of its special structure, the energies of base stackings are not
as important for i-motifs as for B-DNA double helices. The enthalpy effects are usually opposed by
entropic effects, since the entropy of loose DNA strands is lowered when secondary structures are
formed. In most cases, in biologically relevant systems both effects compensate each other for the
most part, ensuring that the overall reaction stays reversible, which means, that the Gibbs energy is
kept close to zero (Searle und Williams 1993). Determining the thermodynamic parameters of the imotif forming structures analyzed in this work may provide insight in the influence of the sequence
variations on the structure and the forces contributing to the stability of each sequence.
The van’t Hoff plots (ln(K) versus 1/T) of I3 for pH 5.1 and 5.4 are shown in Fig. 31.
49
— Results and discussion —
Fig. 31: Van’t Hoff plots (ln (K) vs. 1/T) for I3 at pH 5.1 and pH 5.4. The corresponding melting
temperatures (the temperature where K=1 and thus ln(K)=0) are marked by vertical lines. Linear plots
were applied to the data, R² values are given.
The calculated thermodynamic parameters are summarized in Tab. 11.
Tab. 11: Thermodynamic parameters calculated for I3 at pH 5,1 and 5,4.
sequence
pH
Tm [°C]
(± 0.1)
ΔH°
[kcal/mol]
ΔS°
[cal/K ∙ mol]
ΔG (37°C)
[kcal/mol]
ΔG (25°C)
[kcal/mol]
R²
I3
5.1
48.1
-44.15 ± 0.40
-137.39 ± 1.23
-1.54 ± 0.21
-3.19 ± 0.42
0.993
I3
5.4
40.5
-43.51 ± 0.50
-138.92 ± 1.60
-0.42 ± 0.06
-2.09 ± 0.32
0.985
The R² values show that the two-state model fits to the obtained data, so again the hypothesis of an
intramolecular folding is strengthened. Gibbs energies were calculated for two temperatures, namely
37°C as physiological temperature and 25°C since this is the temperature at which the CD spectra
from section 4.1 were recorded. The values for 37°C (-1.54°C ± 0.21 for pH 5.1 and -0.42°C ± 0.06 for
pH 5.4) are indeed close to zero and thus close to the equilibrium, as it is typical for physiological
processes. The values for 25°C are about 1.5 kcal/mol lower for both pH values, which confirms that
I3 is stabilized by lower temperatures. As predicted above, the enthalpy is negative due to favorable
base stackings and hydrogen bondings; the entropy is negative because folding reduces the levels of
freedom the molecule has access to. For the higher pH, both enthalpic and entropic input to the
Gibbs energy get less favorable compared to the values for pH 5.1 (the enthalpy is less negative, the
entropy is more negative).
Various values for all these three parameters have been reported: Leroy et al. calculated
ΔH° = -59.95 kcal/mol and ΔS = -188 cal/(K∙mol) at pH 6.4 for the same sequence (Leroy et al. 1994),
which
means
ΔG = -1.64 kcal/mol
at
37°C.
Mergny
et
al.
published
values
of
50
— Results and discussion —
ΔG (37°C) = -0.45 kcal/mol, ΔH° = -67 kcal/mol and ΔS° = -215 cal/(K∙mol) for pH 6.0 (Mergny et al.
1995). So actually the obtained values seem to be a bit too low, since it is expected that the Gibbs
energy gets lower when the pH draws near to pKa (but this is not the case for the quoted values,
too).
51
— Results and discussion —
4.3.2. I4 and I6: effect of elongation of the cytosine-rich tracts on the thermal stability
Melting curves for I4 and I6, both at pH 5.1 and 5.4, were measured to monitor the effect of the
additional cytidines in the c-rich tracts on the melting temperature and on the thermodynamic
parameters. Mergny et al. analyzed oligonucleotides containing four cytidine tracts with different
numbers, where (C2T3)3C2 had a melting temperature of 27°C, (C3T3)C3 one of 45°C, for (C4T3)C4 it was
54°C and for (C5T3)C5 59.5°C, so every tract elongation caused a shift to higher melting temperatures,
but not linearly (for all the measurements, the pH was 6.0) (Mergny et al. 1995). Determining the
thermodynamic parameters from the melting curve data may provide information about structural
changes due to the additional base pairs. Also it is interesting to observe whether the pH has an
increased or decreased effect on the melting temperature and thermal stability on a structure with
more hemiprotonated C∙C+ base pairings.
Determination of melting temperatures
The melting curves of I4 and I6 at pH 5.1 are, together with the one of I3 already described in section
3.5.1, shown in Fig. 32.
Fig. 32: Melting curves of I3, I4 and I6 at pH 5.1. The measured ellipticity values were converted into the
ratio of folded to unfolded DNA. Sigmoidal fits were attached to the data; the melting temperature, which
is defined as the temperature at the transition point of the curve, is marked by vertical lines. R²
parameters for the fits are given.
Both I4 and I6 show cooperativity as I3 does. For I4 a melting temperature of 62.7°C ± 0.04, for I6 of
72.8°C ± 0.09 is observed. So to the melting temperature of I3 (ca. 48.1°C ± 0.07), there is a
difference of ΔTm = 14.6°C ± 0.11 for I4 and of ΔTm = 24.7°C ± 0.16 for I6. The difference in the
melting temperatures between I4 and I6 is therefore about 10°C. Thus the shift in melting
temperature is larger going from I3 to I4 than from I4 to I6, although in the latter step four additional
cytidines are introduced that may form hydrogen bonds among themselves. So most likely, an
52
— Results and discussion —
entropic effect is responsible. Further inside should provide the calculation of the thermodynamic
parameter, but first the melting curves of I3, I4 and I6 at pH 5.4 are presented (Fig. 33).
Fig. 33: Melting curves of I3, I4 and I6 at pH 5.4. The measured ellipticity values were converted into the
ratio of folded to unfolded DNA. Sigmoidal fits were attached to the data; the melting temperature, which
is defined as the temperature at the transition point of the curve, is marked by vertical lines. R²
parameters for the fits are given.
Again cooperativity can be detected for the thermal denaturation of each sequence. Each melting
temperature is, as it was expected and already observed for I3, shifted to lower temperature ranges
(about 8°C) due to destabilization by the lower H+-concentration. The difference between the melting
points of I3 and I4 is ΔTm = 13.5°C ± 0.08, between I3 and I6 ΔTm = 23.6°C ± 0.11 and between I4 and
I6 ΔTm = 10.1°C ± 0.11 – in comparison to the values obtained for pH 5.1, the differences between Tm
for I3 and another sequence is for both reduced by 1°C, whereas the difference between the values
for I4 and I6 stays exactly the same. This may be a hint that for these two sequences the pH range
where the melting temperature is linearly correlated with the pH is reached because of a higher
stability against pH changes. For I3, this was negated in section 4.3.1.
The fitting parameters for both melting curves are given in Tab. 12.
Tab. 12: Fitting parameter for the two-state model fits for I4 and I6 at pH 5.1 and 5.4.
sequence
pH
A1
A2
logx0
p
R²
I4
5.1
0.015 ± 0.002
0.959 ± 0.001
62.689 ± 0.041
-0.144 ± 0.002
0.998
I4
5.4
0.022 ± 0.001
0.953 ± 0.001
54.009 ± 0.040
-0.156 ± 0.002
0.998
I6
5.1
0.013 ± 0.005
0.938 ± 0.020
72.811 ± 0.085
-0.148 ± 0.004
0.991
I6
5.4
0.089 ± 0.003
0.927 ± 0.002
64.100 ± 0.075
-0.178 ± 0.005
0.992
53
— Results and discussion —
The hill coefficients p show that for both sequences the cooperativity of the unfolding is slightly
enhanced for the melting curve that was recorded at the higher of both pH vaules. This increase is
larger for I6 than for I4 and almost equal to the one that was observed for I3.
Determination of thermodynamic parameters
From the thermal melting curves, thermodynamic parameters were calculated. The corresponding
van’t Hoff plots for pH 5.1 and pH 5.4 are given in Fig. 34 and Fig. 35.
Fig. 34: Van’t Hoff plots (ln (K) vs. 1/T) for I3, I4 and I6 at pH 5.1. The corresponding melting temperatures
(the temperature where K=1 and thus ln(K)=0) are marked by vertical lines. Linear plots were applied to
the data, R² values are given.
Fig. 35: Van’t Hoff plots (ln (K) vs. 1/T) for I3, I4 and I6 at pH 5.4. The corresponding melting temperatures
(the temperature where K=1 and thus ln(K)=0) are marked by vertical lines. Linear plots were applied to
the data, R² values are given.
The obtained thermodynamic parameters are presented in Tab. 13.
54
— Results and discussion —
Tab. 13: Calculated thermodynamic parameters for I3, I4 and I6 at pH 5,1 and 5,4.
sequence
pH
Tm [°C]
(± 0.1)
ΔH°
[kcal/mol]
ΔS°
[cal/K ∙ mol]
ΔG (37°C)
[kcal/mol]
ΔG (25°C)
[kcal/mol]
R²
I3
5.1
48.1
-44.15 ± 0.40
-137.39 ± 1.23
-1.54 ± 0.21
-3.19 ± 0.42
0.993
I3
5.4
40.5
-43.51 ± 0.50
-138.92 ± 1.60
-0.42 ± 0.06
-2.09 ± 0.32
0.985
I4
5.1
62.7
-69.35 ± 0.98
-206.80 ± 2.92
-5.22 ± 0.80
-7.70 ± 1.18
0.985
I4
5.4
54.0
-70.56 ± 0.91
-215.91 ± 2.79
-3.60 ± 0.54
-6.19 ± 0.93
0.989
I6
5.1
72.8
-89.05 ± 1.63
-257.67 ± 4.69
-9.13 ± 1.52
-12.22 ± 2.04
0.980
I6
5.4
64.1
-69.57 ± 1.45
-206.29 ± 4.28
-5.58 ± 0.97
-8.06 ± 1.40
0.969
The R² values show that it is far more difficult to obtain a smooth fitting for the van’t Hoff plot, which
is a result of the way of the plotting (because of plotting 1/T instead of T, little variability in T gets
very large). Not surprisingly, the Gibbs energies are more favorable for the folding at 25°C than at
37°C, as well as for the lower pH. Indeed, as it was expected, ΔG increases from I3 over I4 to I6. The
differences in ΔG for I4 are at both pH about 2.5 kcal/mol, 1 kcal/mol more than it was observed for
I3. For I6 at pH 5.1, it is close to 3 kcal/mol, at pH 5.4 about 2.5 kcal/mol again; so the Gibbs energy
appears to be more temperature dependent for I4 and I6 than for I3. This can also be seen in the
more rapid decrease of ΔG at pH 5.1 from I3 over I4 to I6 at 37°C than at 25°C. The differences in ΔG,
comparing the values for each sequence at both pH, are for I4 about 1.5 kcal/mol at both
temperatures, for I6 at 37°C it is about 3 kcal/mol, at 25°C about 2.5 kcal/mol. Since for I3 it had been
1 kcal/mol, there is a correlation between the length of the cytidine stretch and the pH-dependency
of ΔG, too. But whereas for I4 only one magnitude seems to influence the Gibbs energy at the same
time (that means, the differences in ΔG according to temperature stay the same at a fixed pH, as well
as it remains constant when the temperature is fixed and the pH is varied), both temperature and pH
have influence on the Gibbs energy of unfolding for I6.
The effect of introducing cytidines in the sequence does here not result in a linear stabilization, in
agreement with the findings of Mergny et al. (1995). At pH 5.1 and 37°C, the difference in ΔG is
about 3.5 kcal/mol, comparing I3 and I4, but only 4 kcal/mol for I4 and I6, not 7 kcal/mol as a
linearity would require. At pH 5.4 the Gibbs energy difference is even smaller for I4 and I6 than for I3
and I4. The same is true for the values calculated for a temperature of 25°C; it is interesting to note
that the gain in free energy at 25°C is larger, comparing I3 and I4, than at 37°C, whereas for I6 it is the
other way round. Since the Gibbs energy is composed of contributions from enthalpy and energy, it is
important to discuss them as well:
At pH 5.1, the enthalpy of I4 is about 25 kcal/mol lower than of I3, and the one of I6 is additional
20 kcal/mol lower (to a total difference of 45 kcal/mol from I3 to I6). At pH 5.4, instead, the energy
55
— Results and discussion —
gain is about the same amount between I3 and I4, but from I4 to I6 it stays, taking the error into
account, the same. So as the enthalpy difference within the sequences is similar for the different pH,
the change from pH 5.1 to 5.4 causes an enthalpy alteration of almost 20 kcal/mol to less
favorableness. The same can be observed for the entropy (except that the elongation here causes a
decrease in entropy, that means less favorableness): enhancing the pH causes an increase in negative
entropy with a large difference comparing I3 and I4 (ca. 70 cal/(K ∙ mol)) and I4 and I6 (another
50 cal/(K ∙ mol)) – but only for pH 5.1, since at pH 5.4 the entropy gets more favorable for I6, even
more favorable than the one of I4, while the entropy change of 70 cal/(K ∙ mol) comparing I3 and I4
remains. So in the transition from pH 5.1 to 5.4, something is happening which causes the enthalpy
to get less favorable, but the entropy to get more favorable. Searle et al. explain the Gibbs energy of
nucleotides as a result of the compensation of a large enthalpy gain due to base stacking interactions
and hydrogen bondings by a large entropy-based amount of unfavorable free energy which is caused
by reduction of degrees of freedom by ordering the phosphate backbone of the nucleotide (Searle
und Williams 1993). This principle can be observed very well in the obtained data for the elongated
oligonucleotides, leaving I6 at pH 5.4 out: more nucleotides, more enthalpy gain, and more entropy
loss. Corresponding to this, the explanation for the odd behavior of the I6 must lie in a partial
unfolding of the structure which restores some degrees of freedom and dissolves some base stacking
interactions. But this explanation does not go along with the recorded melting curve, which clearly
shows a cooperative unfolding; it seems tempting to assume a cooperative unfolding behavior due to
heating, but uncooperative behavior when exposed to a pH increase.
To sum the results of this section up, a few conclusions can be drawn: the observed i-motifs were
stabilized both by low temperature and by low pH. The thermal induced unfolding of all was
cooperative. Adding cytidines to the c-rich stretches caused a shift in melting temperature to higher
temperatures, but no linear coherence between the number of hemiprotonated cytidine-cytidine
base pairs and the shift in melting temperature was observed. For I6, there are hints for a different
behavior as reaction to an increase of temperature than to an increase of pH.
56
— Results and discussion —
4.3.3.
I3Me2 and I3Me4: effect of 5N-methylation of cytidine on the thermal stability of
i-motifs
As described in the section concerning the CD spectrometric measurements, methylations were
shown to stabilize triplex DNA against both pH and temperature increase (Lee et al. 1984) (Xodo et
al. 1991). The effect of methylations on the i-motif has been reported little yet; Mergny et al.
compared the melting temperature of d(TC2TC2T4C2TC2T) with that of a DNA strand with the same
sequence, but all cytidines substituted by 5-methylcytidine. No significant difference (±2°C) in the
melting temperature was measured for the two sequences in a pH range from 4.4 to 7.2.
Nevertheless, although the Gibbs energy stayed similar, changes in enthalpy and entropy between
the methylated and not methylated sequence were calculated, so the methylations definitely had an
effect on the i-motif (Mergny et al. 1995). For this reason the melting temperature and
thermodynamic parameters of I3Me2 and I3Me4 were determined here as well.
Determination of melting temperatures
Fig. 36 shows the curves obtained from the CD melting experiments on I3, I3Me2 and I3Me4 at pH
5.1.
Fig. 36: Melting curves of I3, I3Me2 and I3Me4 at pH 5.1. The measured ellipticity values were converted
into the ratio of folded to unfolded DNA. Sigmoidal fits were attached to the data; the melting
temperature, which is defined as the temperature at the transition point of the curve, is marked by
vertical lines. R² parameters for the fits are given.
57
— Results and discussion —
For all three sequences sigmoidal fits with good R²values can be attached to the data, suggesting that
the temperature induced unfolding of the i-motifs is cooperative. The methylations seem to have a
stabilizing effect on the structure, but only a slight one: the difference in melting temperature
(Tm)between I3 and I3Me2 is about 2.5°C, the one between I3 and I3Me4 about 4.5°C (so among
themselves ΔTm = 2°C). The difference is too small to make a statement about coherence of melting
temperature and number of methyl substitutions in the sequence.
The melting curves obtained at pH 5.4 are given in Fig. 37.
Fig. 37: Melting curves of I3, I3Me2 and I3Me4 at pH 5.4. The measured ellipticity values were converted
into the ratio of folded to unfolded DNA. Sigmoidal fits were attached to the data; the melting
temperature, which is defined as the temperature at the transition point of the curve, is marked by
vertical lines. R² parameters for the fits are given.
These curves reveal also a cooperative temperature induced unfolding of the structures. Again the
difference in the melting temperatures is small: ΔTm = 1.291°C ± 0.09 concerning I3 and I3Me2 and
ΔTm = 3.776°C ± 0.113 for I3 and I3Me4 (for I3Me2 and I3Me4 ΔTm = 2.485°C ± 0.107). So the
decrease of the melting temperature due to the increased pH seems to be higher for I3Me2 than for
I3Me4 and no linearity can be detected.
The fitting parameters for both sequences and pH are listed in Tab. 14.
Tab. 14: Fitting parameters for the two state model fits for I3Me2 and I3Me4 at pH 5.1 and 5-4.
sequence
pH
A1
A2
logx0
p
R²
I3Me2
5.1
0.020 ± 0.002
0.955 ± 0.002
50.646 ± 0.063
-0.089 ± 0.001
0.998
I3Me2
5.4
0.027 ± 0.001
0.961 ± 0.002
41.785 ± 0.048
-0.096 ± 0.001
0.999
I3Me4
5.1
0.032 ± 0.002
0.938 ± 0.002
52.530 ± 0.078
-0.092 ± 0.001
0.996
I3Me4
5.4
0.031 ± 0.002
0.952 ± 0.002
44.270 ± 0.065
-0.095 ± 0.001
0.997
58
— Results and discussion —
The difference between the melting temperatures of each sequence at the different pH values
(pH 5.1 and 5.4) is about 8.8°C for I3Me2 and 8.2°C for I3Me4. For I3, a difference of 7.7°C has been
observed. No direct correlation can be derived from here. The cooperativity of the unfolding, which
can be deduced from the hill coefficient p of the sigmoidal fit, seems to be for pH 5.1 slightly higher
for both I3Me2 and I3Me4 than for I3 (where a hill coefficient of -0.084 ± 0.001 was obtained) and
slightly lower for pH 5.4 (-0.106 ± 0.001 for I3).
59
— Results and discussion —
Determination of thermodynamic parameters
For further inside, the thermodynamic parameters were calculated from the melting curve data. The
van’t Hoff plots of I3, I3Me2 and I3Me4 at pH 5.1 are given in Fig. 38, at pH 5.4 in Fig. 39.
Fig. 38: Van’t Hoff plots (ln (K) vs. 1/T) for I3, I3Me2 and I3Me4 at pH 5.1. The corresponding melting
temperatures (the temperature where K=1 and thus ln(K)=0) are marked by vertical lines. Linear plots
were applied to the data, R² values are given.
Fig. 39: Van’t Hoff plots (ln (K) vs. 1/T) for I3, I3Me2 and IeMe4 at pH 5.4. The corresponding melting
temperatures (the temperature where K=1 and thus ln(K)=0) are marked by vertical lines. Linear plots
were applied to the data, R² values are given.
The fittings for the curves obtained from the measurements at pH 5.1 look almost parallel to each
other, so it is predicted that here the ΔH° values for the different sequences are similar and only a
difference in ΔS° is observable. The curves obtained at pH 5.4 instead will have differences in both
enthalpy and entropy. The calculated thermodynamic parameters are summarized in Tab. 15.
60
— Results and discussion —
Tab. 15: Calculated thermodynamic parameters for I3, I3Me2 and I3Me4 from the melting curves measured at pH 5.1 and
5.4.
sequence
pH
Tm [°C]
(± 0.1)
ΔH°
[kcal/mol]
ΔS°
[cal/K ∙ mol]
ΔG (37°C)
[kcal/mol]
ΔG (25°C)
[kcal/mol]
R²
I3
5.1
48.1
-44.15 ± 0.40
-137.39 ± 1.23
-1.54 ± 0.21
-3.19 ± 0.42
0.993
I3
5.4
40.5
-43.51 ± 0.50
-138.92 ± 1.60
-0.42 ± 0.06
-2.09 ± 0.32
0.985
I3Me2
5.1
50.6
-43.65 ± 0.46
-134.96 ± 1.43
-1.79 ± 0.25
-3.41 ± 0.48
0.989
I3Me2
5.4
41.8
-41.53 ± 0.52
-132.00 ± 1.60
-0.59 ± 0.09
-2.17 ± 0.32
0.987
I3Me4
5.1
52.5
-45.24 ± 0.55
-139.00 ± 1.67
-2.13 ± 0.31
-3.80 ± 0.56
0.990
I3Me4
5.4
44.3
-38.36 ± 0.44
-120.12 ± 1.39
-1.11 ± 0.16
-2.55 ± 0.37
0.993
The thermodynamic parameters indeed show the characteristics that were predicted: The enthalpy is
for I3 and I3Me2 at both pH values plus I3Me4 at pH 5.1 almost the same, whereas it drops about
7 kcal/mol for I3Me4 comparing pH 5.1 and 5.4 to less favorableness. The entropy shows the same
tendency, apart from a rise for I3Me4 from pH 5.1 to 5.4, so the free energy loss in enthalpy is
compensated to some extent by a more favorable entropy. Similar had already been shown for the I6
sequence (see 3.5.2.).The change from pH 5.1 to pH 5.4 results in a change of about 1.2 kcal/mol in
Gibbs energy for I3Me2 at 37°C and of ca. 1.6 kcal/mol at 25°C. For I3Me4 the same effect is
observed: here ΔΔG is close to 1 kcal/mol in the transition from pH 5.1 to 5.4 at 37°C and
1.4 kcal/mol at 25°C. So it seems that the methylations do not only slightly stabilize the i-motif, but
also reduce the temperature dependency of the Gibbs energy. For I4 and I6, the contrary has been
observed. Varying the temperature, not the pH, leads to ΔΔG values of about 1.6 kcal/mol more
stabilization for I3Me2 at pH 5.1 and 1.6 kcal/mol at pH 5.4 as well. For I3Me4, these differences are
about 1.7 kcal/mol at pH 5.1 and slightly reduced to 1.4 kcal at pH 5.4. This may be a hint for a
greater pH-dependency of the Gibbs energy for I3Me4 than for I3Me2. Definitely the effect of the
fourfold methylation is greater than the effect of the twofold methylation of I3, but simultaneously
the pH seems to gain influence on the stability of the structure. Mergny et al. observed for their
comparison
of
the
thermodynamic
data
on
d(TC2TC2T4C2TC2T)
and
d(T(5mC)2T(5mC)2T4(5mC)2T(5mC)2T) a constant lowering of enthalpy, entropy and melting
temperature from pH 5.6 over 6.4 to 6.8 for the not methylated structure. For the methylated
sequence instead there was an enthalpy change comparing pH 5.6 and 6.4 to less favorable energies
accompanied by a change to more favorable entropies (Mergny et al. 1995). This is similar to the
observations on I3Me4 for the transition from pH 5.1 to 5.4 that were made here. Except for this, the
enthalpy stayed more or less the same for pH 5.6, 6.4 and 6.8 for both methylated and not
methylated sequence (48 kcal/mol with a difference of ±2-3 kcal/mol) (Mergny et al. 1995) as it was
observed here, too. However, the effects are only small. It would be interesting to investigate if these
61
— Results and discussion —
findings can be conserved if more methylations are introduced and whether this increases the pH
influence as well.
To sum up the observations that were discussed in this section: The unfolding induced by increase of
temperature showed cooperativity for both sequences. The melting temperature for both I3Me2 and
I3Me4 are, compared to the one of I3, increased. The effect is small compared to the one observed
for I4 and I6.
62
— Results and discussion —
4.3.4. I3Br2 and I3Br4: effect of 5N-bromination on the thermal stability of the i-motifs
The influence of a bromine-substitution on the thermal stability of the i-motif has not been reported
so far. The pH response studies showed a large destabilization against basic pH. With the
determination of the melting temperature it is shown here that the thermal stability is reduced as
well. Since it was stated in the introduction to the section on the CD measurements of I3Br2 and
I3Br4 that the effect of the bromine substitutions would be mostly based on an influence on the pKa
of cytidine and therefore on the hydrogen bonding between the hemiprotonated base pairs, an
analyses of the thermodynamic parameters may reveal if the loss in stabilization is more due to a rise
in enthalpy, loss of entropy or both.
Determination of the melting temperature
Fig. 40 shows the melting curves that were recorded for I3, I3Br2 and I3Br4 at pH 5.1.
Fig. 40: Melting curves of I3, I3Br2 and I3Br4 at pH 5.1. The measured ellipticity values were converted
into the ratio of folded to unfolded DNA. Sigmoidal fits were attached to the data; the melting
temperature, which is defined as the temperature at the transition point of the curve, is marked by
vertical lines. R² parameters for the fits are given.
All three curves show a cooperative unfolding of the structure due to the temperature decrease. The
melting temperature is shifted to lower temperatures for I3Br2 compared with I3 and another
reduction is observable between I3Br2 and I3Br4. The temperature differences between the
sequences are about 9.3°C for I3 and I3Br2 and 18.4°C for I3 and I3Br4, so maybe the reduction of
the melting temperature is correlated linearly with the number of brominations in the molecule.
Since bromine reduces the pKa of cytidine, as it was shown in the pH-response range determinations,
it is assumable that the pH 5.1 and 5.4 are in the pH range of linear coherence of pH and melting
temperature, which means, not close to the pKa.
The data from the measurements on I3, I3Br2 and I3Br4 at pH 5.4 is plotted in Fig. 41.
63
— Results and discussion —
Fig. 41: Melting curves of I3, I3Br2 and I3Br4 at pH 5.4. The measured ellipticity values were converted
into the ratio of folded to unfolded DNA. Sigmoidal fits were attached to the data; the melting
temperature, which is defined as the temperature at the transition point of the curve, is marked by
vertical lines. R² parameters for the fits are given.
The curves show a sigmoidal unfolding behavior as well. The melting temperatures are again lower
for I3Br2and I3Br4 than for I3. It should be noticed here that the fitting for I3Br4 was influenced
insofar the parameter for the upper asymptote was fixed at 1, because otherwise a fitting wouldn’t
have been successful due to the unfolding at even low temperatures (to correct this the melting
curve should have been recorded with a lower starting temperature). So it is possible that the
melting temperature stated here is false and might be even lower. There is a temperature difference
about 8.8°C between I3 and I3Br2 and one of 9.9°C (or more) between I3Br2 and I3Br4, so here is no
hint for a coherence to the number of bromine substitutions detectable.
The fitting parameters for all sigmoidal fits attached to the melting curve data of I3Br2 and I3Br4 are
listed in Tab. 16.
Tab. 16: Fitting parameters for the two state model fits for I3Br2 and I3Br4 at pH 5.1 and .,4.
sequence
pH
A1
A2
logx0
p
R²
I3Br2
5.1
0.044 ± 0.001
0.945 ± 0.002
38.816 ± 0.067
-0.092 ± 0.001
0.997
I3Br2
5.4
0.032 ± 0.002
0.993 ± 0.002
31.708 ± 0.048
-0.082 ± 0.048
0.999
I3Br4
5.1
0.083 ± 0.002
0.973 ± 0.005
29.746 ± 0.118
-0.077 ± 0.001
0.993
I3Br4
5.4
0.090 ± 0.002
1±0
21.805 ± 0.111
-0.074 ± 0.001
0.985
The hill coefficients show, compared with the ones of I3 (-0.084 ± 0.001 at pH 5.1 and -0.106 ± 0.001
at pH 5.4) that the cooperativity of the unfolding is maintained mostly for I3 and I3Me2, whereas the
cooperativity for I3Br4 compared to that of I3 is, particularly at pH 5.4, reduced. Similar behavior was
observed for the pH responses.
64
— Results and discussion —
Because the melting temperatures on their own provide little information, the thermodynamic
parameters are determined from the melting curves.
Determination of thermodynamic parameters
The van’t Hoff plots for I3, I3Br2 and I3Br4 at pH 5.1 and 5.4 are shown in Fig. 42 and Fig. 43.
Fig. 42: Van’t Hoff plots (ln (K) vs. 1/T) for I3, I3Br2 and I3Br4 at pH 5.1. The corresponding melting
temperatures (the temperature where K=1 and thus ln(K)=0) are marked by vertical lines. Linear plots
were applied to the data, R² values are given.
Fig. 43: Van’t Hoff plots (ln (K) vs. 1/T) for I3, I3Br2 and I3Br4 at pH 5.4. The corresponding melting
temperatures (the temperature where K=1 and thus ln(K)=0) are marked by vertical lines. Linear plots
were applied to the data, R² values are given.
The mere appearance of the linear curves leads to the prediction that the differences of the
thermodynamic parameters between I3Br2 and I3Br4 are not as important as between I3 and the
brominated sequences. The calculated values are given in Tab. 17.
65
— Results and discussion —
Tab. 17: Calculated thermodynamic parameters for I3, I3Br2 and I3Br4 from the melting curves measured at pH 5.1 and
5.4.
sequence
pH
Tm [°C]
(± 0.1)
ΔH°
[kcal/mol]
ΔS°
[cal/K ∙ mol]
ΔG (37°C)
[kcal/mol]
ΔG (25°C)
[kcal/mol]
R²
I3
5.1
48.1
-44.15 ± 0.40
-137.39 ± 1.23
-1.54 ± 0.21
-3.19 ± 0.42
0.993
I3
5.4
40.5
-43.51 ± 0.50
-138.92 ± 1.60
-0.42 ± 0.06
-2.09 ± 0.32
0.985
I3Br2
5.1
38.8
-28.73 ± 0.19
-91.42 ± 0.61
-0.38 ± 0.05
-1.47 ± 0.19
0.995
I3Br2
5.4
31.7
-33.10 ± 0.22
-108.42 ± 0.73
0.53 ± 0.07
-0.76 ± 0.10
0.996
I3Br4
5.1
29.7
-30.86 ± 0.30
-101.65 ± 1.09
0.67 ± 0.09
- 0.55 ± 0.08
0.989
I3Br4
5.4
21.8
-29.49 ± 0.47
-99.65 ± 1.58
1.42 ± 0.23
0.22 ± 0.04
0.979
Consistent with what was predicted, the bromine substitutions influence both entropy and enthalpy,
while both together result in a less favorable Gibbs energy for I3Br2 and I3Br4 at 25°C. I3Br4 has a
negative Gibbs energy at 25°C and pH 5.1, which is close to the equilibrium, and a positive one at
25°C and pH 5.4, which means that the consideration of a partly unfolded I3Br4 in the CD
spectrometric measurements is not correct: at pH 5.1 the structure seems to be folded, but the
unfolding will start at pH values that are only a little higher. The particular differences in Gibbs
energy that result from the pH change from pH 5.1 to 5.4 for both sequences and both temperatures
are almost equal, so the Gibbs energy is more or less independent of pH as well as of temperature in
the ranges that are investigated here. At 37°C, even I3Br2 at pH 5.4 has a positive Gibbs energy and is
therefore unfolded.
Comparing I3 and I3Br2, the effect on the enthalpy seems to be higher for pH 5.1 than for pH 5.4,
because the energy differences are about 16 kcal/mol at pH 5.1 to a less negative value, but only
10 kcal/mol at pH 5.4. For I3Br4, the enthalpy at the different pH values is almost the same
(± 2 kcal/mol). Actually the effect on the entropy is small as well, adding up to only slightly different
Gibbs energies. For both sequences the entropy is less negative than for I3, which means more
favorable. If it was not for the loss of negative enthalpy, an explicit stabilizing effect should be
observable. So maybe the bromines disturb the intercalation of the bases, which would lead to the
reduction of favorable enthalpy but also to the increase in entropy. Maybe in a structure containing
both methylations and brominations it would be possible to combine the enthalpy maintaining effect
of the methyl substitutions with the entropy increasing effect of the bromine substitution and create
an i-motif with a greater stability at 37°C.
All in all, both I3Br2 and I3Br showed lower melting temperatures at both pH values than I3. The
Gibbs energy was less favorable as well, due to both higher enthalpies and lower entropies.
66
— Conclusion and outlook —
5.
Conclusion and outlook
In this bachelor thesis, the i-motif forming capacity, the pH-response range and the thermal stability
of c-rich structures, all derived from the human telomere fragment d(C3TA2)C3 (“I3”), were analyzed.
The ambition was to monitor the changes in these properties and connect them to the sequence
variations that have been made. This should lead to the possibility to design new i-motif based
nanodevices with a broader pH-response range or pH-response ranges that are shifted to more acidic
or basic pH ranges than the i-motif based nanodevices invented so far.
5.1.
pH-response range
The recording of the CD spectra revealed that all sequences that were used here can fold into an imotif. The pH-response range of I4, the sequence with four cytidines in each c-rich tract (instead of
three as in I3) broadened about 0.2 pH units in both directions (more acidic and more basic) from
pH 5.8 – 6.2, the pH-response range of I3, to pH 5.4 – 6.6. But the results have not been explicit
regarding the folding mechanism of the structure. The fitting for an assumed three-state model, i.e. a
folding that includes a folding intermediate, matches much better to the data than the fitting
corresponding to a two-state model. For I6, the fitting procedure was even more complicated and
allows no statement about the folding mechanism or the pH-response range. It may be possible that
the pH range is broadened in such a manner that it was not possible to observe it in the pH range
that was tested here.
For I3Me2, the sequence in which two cytosines in the middle of the c-rich tract of I3 were
substituted by 5-methylcytosines, the pH-response range was broadened a little and its transition
point was shifted about ΔpH = 0.1 to more basic pH. For I3Me4, another slight transition point shift
to higher pH values could be observed, but here the pH-response range was narrowed. The
assumption that methylations of cytidine would cause a pH shift to more basic pH (because the pKa
of 5-methylcytidine is slightly higher than the one of cytidine) was therefore confirmed.
For the bromine-substituted cytosines it was expected that the pH-response range is shifted towards
more acidic pH values, because 5-bromocytosine exhibits a lower pKa than cytosine. This was shown
for both I3Br2 and I3Br4: the pH transition point of I3Br2 is shifted about 0.3 pH units to a pH of 5.7,
the one of I3Br4 is at pH 5.1. Additionally, the pH range of I3Br4 is broadened slightly.
Following, regarding the pH-response of I4 and I6 it would be necessary to get evidence about the
actual folding mechanism. For I6 the first step would be to make measurements with a higher
concentration of DNA and to add samples both in the lower and higher pH range. This may confirm
the hypothesis that all the pH values that were measured here lie in the transition part of the pHresponse curve. For I4 on the other hand it would be useful to define the transitional part to improve
the fitting. The effect of the methylations is indeed very small, maybe too small to be of use for a
67
— Conclusion and outlook —
possible nanodevice. But besides a broadening of the pH range, a narrow pH-response range may be
useful as well, for example for some process were the pH has to be determined very accurate. The
effect of the brominations was much more important and is possibly applicable to monitor activities
in very acidic cell compartments where other nanodevices are not of use because of the high H+concentration. It would be interesting to combine the sequence variations, for example extract the
cytidine-tracts and introduce methylations at the same time. Maybe it is possible to combine the
different effects on the pH-response range that were observable here. Besides it is considerable that
a possible I5 structure with five cytidines one after another in the c-rich tracts would have the
desired effects on the pH response.
5.2.
Stoichiometry of the i-motifs
For all of the sequences that were analyzed here, an intramolecular folding of the i-motif in a native
polyacrylamide gel at pH 5.0 was proven.
5.3.
Thermal stability
Besides the pH-response, the thermal stability was analyzed by CD melting curve measurements. For
each sequence, melting curves at pH 5.1 and 5.4 were recorded. The determining of Gibbs energy
ΔG, enthalpy ΔH and entropy ΔS should provide insight about the effect of the sequence alterations
on the stability of the i-motif.
The melting curves showed a cooperative unfolding mechanism for the temperature induced
unfolding of the i-motifs for all sequences. The dependency of the melting temperature on pH was
observed for all sequences as well. For I4 and I6, a higher melting temperature was observed than for
I3, but the effect was larger comparing I3 and I4 than for I4 and I6, which means either that the
stabilization is not linearly correlated to the number of C∙C+ base pairs or that for I6 not all the
cytidines bases are incorporated into base pairing. For all sequences an increase of temperature as
well as the higher pH (pH 5.4) caused an increase of Gibbs energy to less favorableness. For I4 and I6,
the additional cytidines had a favorable effect on the enthalpy, but accompanied by a unfavorable
loss in entropy. Herein, the thermodynamic parameters of I6 revealed a much larger pH and
temperature dependency than the ones of I4. The substitution of cytosine by 5-methylcytosine,
again, had only little effect; the Gibbs energies of I3 and I3Me2 at pH 5.4 are almost equal at 37°C.
I3Br2 and I3Br4 at last showed a great shift in the melting temperature compared to I3, namely to
the lower temperature range. The measurements revealed that indeed for both pH (pH 5.1 and 5.4)
the Gibbs energies are close to the equilibrium. At 37°C, I3Br4 is unfolded at pH 5.1 as well as at
pH 5.4. At 25°C, the Gibbs energy is negative at pH 5.1, but not at pH 5.4. These results show that, in
order to find a possible nanodevice applicable to biological systems, the pH range measurements had
better been measured at 37°C, because stability of the i-motifs is affected by temperature as well.
68
— Conclusion and outlook —
Indeed, the shift of the pH-response range of I3Br2 and I3Br4 to lower pH regions that was
considered useful for measurements in acidic cellular compartments above seems now mostly
worthless, because the temperature has such an destabilizing effect. To gain more insight in the
effects of the sequence alterations on the formation and stability of the i-motif, it would be required
to reveal the structure of each, for example by NMR or X-ray crystallography, as it has been already
done for other i-motif structures. Nevertheless it was possible herein not only to prove i-motif
formation for all the sequences that were analyzed here, but also to explain the various effects the
different structure alterations had on the stability of the i-motif formed by d((C3TA2)3C3).
69
— Zusammenfassung —
6.
Zusammenfassung
Die hier präsentierte Arbeit hat sich mit einem Fragment der menschlichen Telomeren-DNA
beschäftigt, die reich an Cytosinen ist. Diese Sequenz, d((C3TA2)C3), kann unter sauren Bedingungen
eine spezielle Sekundärstruktur, das sogenannte „i-motif“, ausbilden. Ein solches i- motif wurde das
erste Mal 1993 von Gehring et al. für die Sequenz d(TC5) beschrieben. Zentrales Merkmal dieser
Struktur ist das hemiprotonierte Cytosin-Cytosin-Basenpaar (C∙C+). Es werden parallele Duplexe
gebildet, die von diesen hemiprotonierten Basenpaaren zusammengehalten werden. Zwei dieser
Duplexe bilden zusammen eine viersträngiges i-motif, indem die beiden Duplexe antiparallel
zueinander angeordnet sind und die Basenpaare vollständig interkalieren (Gehring et al. 1993). Da
für den Aufbau dieser Struktur das hemiprotonierte Basenpaar so essentiell ist, ist das i-motif sehr
pH-sensitiv; sowohl Deprotonierung als auch Protonierung sind ungünstig, weil die Basenpaare
dadurch aufgelöst werden.
Forscher versuchen, sich diese pH-Sensitivität im Bezug auf sogenannte „Nanodevices“ nutzbar zu
machen, Nanometer große molekulare Werkzeuge, mit denen Veränderungen oder Zustände in
biologischen Systemen beobachtet werden können. Sharma et al. beispielsweise haben ein solches
auf dem i-motif basierendes Nanodevice entwickelt, das bei pH 5 und pH 8 unterschiedliche Farben
hat. Der Unterschied zwischen saurem und basischem pH kann also direkt mit dem bloßen Auge
erkannt werden (Sharma et al. 2007). Die meisten Nanodevices auf i-motif-Basis, die bis jetzt
entwickelt wurden, sind aber nur im sauren pH nützlich, weil das i-motif bis auf wenige Ausnahmen
bis jetzt nur unter sauren Bedingungen nachgewiesen werden konnte.
Die vorliegende Arbeit hat sich deshalb mit Derivativen der oben angegebenen Sequenz beschäftigt,
die zum Zwecke einer veränderten pH-Abhängigkeit variiert wurden. Die untersuchten Sequenzen
sind in Tab. 18 aufgelistet.
Tab. 18: C-reiche Sequenzen, die in dieser Arbeit untersucht wurden.
Name
Sequenz (5’-3’)
I3
CCC TAA CCC TAA CCC TAA CCC
I4
CCC CTA ACC CCT AAC CCC TAA CCC C
I6
CCC CCC TAA CCC CCC TAA CCC CCC TAA CCC CCC
I3Me2
CCC TAA C5mCC TAA CCC TAA C5mCC
I3Me4
C5mCC TAA C5mCC TAA C5mCC TAA C5mCC
I3Br2
CCC TAA C5BrCC TAA CCC TAA C5BrCC TAA
I3Br4
C5BrCC TAA C5BrCC TAA C5BrCC TAA C5BrCC
70
— Zusammenfassung —
Bei zweien der Sequenzen wurde jeder der vier cytosinreichen Abschnitte verlängert, im Falle von I4
jeweils um ein Cytosin, bei I6 um drei. Bei I3Me2 und I3Me4 wurden zwei bzw. 4 Cytosine, jeweils
aus der Mitte der C-reichen Abschnitte, gegen 5‘-Methylcytosin ausgetauscht, bei I3Br2 und I3Br4
gegen 5‘-Bromocytosin. Für die Sequenzen mit den verlängerten C-reichen Abschnitte wurde eine
Veränderung der Stabilität gegenüber pH-Änderungen vermutet aufgrund der Ausbildung von mehr
hemiprotonierten C-C-Basenpaaren. Von I3Me2 und I3Me4 wurde erwartet, dass die pH-induzierte
Entfaltung erst bei höheren pH-Werten auftritt, weil die Methylsubstitution den pKa der Cytosine
heraufsetzt, für I3Br2 und I3Br4 sollte der gegenteilige Effekt zu sehen sein, weil der pKa der Cytosine
herabgesetzt ist.
Die Sequenzen wurden zunächst CD-spektroskopisch untersucht. CD-Spektroskopie eignet sich zur
Untersuchung von i-motif-Strukturen, weil diese ein charakteristisches Spektrum ausbilden mit einer
positiven Bande zwischen 285 und 290 nm und einer negativen Bande zwischen 260 und 275 nm. Es
wurden zunächst CD-Spektren von allen Sequenzen bei verschiedenen pH-Werten (zwischen 4,8 und
7,0) aufgenommen, die anschließend in „pH-response range“-Kurven umgewandelt wurden
(Elliptizität bei 288 nm aufgetragen gegen den pH). Über Gelelektrophorese sollte die Stöchiometrie
der i-motif-Faltung bestimmt werde. Im Weiteren wurden noch CD-Schmelzkurven von allen
Sequenzen bei pH 5,1 und pH 5,4 aufgenommen, aus denen thermodynamische Parameter (ΔG, ΔH
und ΔS) berechnet wurden. Diese sollten Aufschluss darüber geben, ob die Veränderungen, die durch
die Variation der Sequenz erzeugt wurden, Einfluss auf entropische und enthalpische Effekte hatte
und ob eine größere oder geringere Stabilität gegenüber Temperaturveränderungen erzeugt wurde.
In Tab. 19 sind die Ergebnisse der CD-spektroskopischen Messungen bezüglich der Stabilität
gegenüber pH-Änderungen dargestellt.
Tab. 19: Zusammenfassung der Ergebnisse, die aus den CD-spektroskopischen Messungen bezüglich der Stabilität
gegenüber pH-Änderungen gewonnen wurden.
Sequenz
pH-response range
Wendepunkt
Kooperativität
verglichen mit I3
I3
5,8 – 6,2
6,0
I4
5,4 – 6,6
6,3
erhöht
I6
?
?
verringert
I3Me2
5,8 – 6,3
6,1
verringert
I3Me4
6,0 – 6,4
6,16
erhöht
I3Br2
5,6 – 6,0
5,7
erhöht
I3Br4
< 4,8 – 5,6
5,1
verringert
Neben den pH-Bereichen, in denen die Entfaltung des i-motifs aufgrund der reduzierten
H+-Konzentration festgestellt wurde, sind der Wendepunkt der Kurve sowie der Vergleich zwischen
71
— Zusammenfassung —
der beobachteten Kooperativität der Entfaltung jeder Sequenz im Vergleich zur Kooperativität der
Entfaltung von I3 zusammengefasst. I4 zeigt eine höhere Kooperativität, aber auch einen größeren
Entfaltungs-pH-Bereich. Es sollte erwähnt werden, dass die erhaltenen Daten keinen Aufschluss über
den Entfaltungsmechanismus von I4 geben konnten; ein Fit, der einen Entfaltungsmechanismus
beschreibt, der ein Intermediat beinhaltet, würde weit besser zu den erhaltenen Daten passen als
der Fit, der nur zwei Populationen (i-motif und ungefaltete Sequenz) zulässt. Es ist aber keine
Alternativstruktur neben dem i-motif bekannt, die über CD-Spektroskopie nachzuweisen wäre. Die
hier angegebenen Werte sind für den Zwei-Populationen-Fit angegeben. Hier wäre also eine
Erweiterung des pH-Bereichs, in dem die Entfaltung stattfindet, zu beobachten. Für I6 indes konnte
überhaupt kein Fit angelegt werden. Die Substitution von Cytosin gegen Methylcytosin hatte nur
einen geringen Effekt, aber wie erwartet wurde der Wendepunkt hin zu größeren pH-Werten
verschoben. Die Bromierungen hatten eine größere Auswirkung, allerdings (wie erwartet) hin zur
Entfaltung bei niedrigeren pH-Werten.
Eine native Polyacrylamid-Gelelektrophorese hat gezeigt, dass alle hier untersuchten Sequenzen ein
intramolekulares i-motif ausbilden. Für I3Br2 und I3Br4 konnten zwei Banden identifiziert werden.
Der Nachweis für die intramolekulare Faltung wurde für I3Br2 über Schmelzkurvenmessungen
nachgewiesen, weil die Schmelztemperatur im Falle einer nicht-intramolekularen Faltung
konzentrationsabhängig
wäre.
Es
konnte
aber
keine
Konzentrationsabhängigkeit
der
Schmelztemperatur festgestellt werden. Für I3Br4 wurde dieses Experiment nicht durchgeführt, weil
zu wenig DNA vorhanden war.
Die Ergebnisse der Schmelzkurven-Messungen für pH 5,1 und 5,4 sind in Tab. 20 angegeben.
72
— Zusammenfassung —
Tab. 20: Zusammenfassung der thermodynamischen Parameter, die aus den Schmelzkurven gewonnen wurden.
Sequenz
pH
Tm [°C]
(± 0.1)
ΔH°
[kcal/mol]
ΔS°
[cal/K ∙ mol]
ΔG (37°C)
[kcal/mol]
ΔG (25°C)
[kcal/mol]
R²
I3
5,1
48,1
-44,15 ± 0,40
-137,39 ± 1,23
-1,54 ± 0,21
-3,19 ± 0,42
0,993
I3
5,4
40,5
-43,51 ± 0,50
-138,92 ± 1,60
-0,42 ± 0,06
-2,09 ± 0,32
0,985
I4
5,1
62,7
-69,35 ± 0,98
-206,80 ± 2,92
-5,22 ± 0,80
-7,70 ± 1,18
0,985
I4
5,4
54,0
-70,56 ± 0,91
-215,91 ± 2,79
-3,60 ± 0,54
-6,19 ± 0,93
0,989
I6
5,1
72,8
-89,05 ± 1,63
-257,67 ± 4,69
-9,13 ± 1,52
-12,22 ± 2,04
0,980
I6
5,4
64,1
-69,57 ± 1,45
-206,29 ± 4,28
-5,58 ± 0,97
-8,06 ± 1,40
0,969
I3Me2
5,1
50,6
-43,65 ± 0,46
-134,96 ± 1,43
-1,79 ± 0,25
-3,41 ± 0,48
0,989
I3Me2
5,4
41,8
-41,53 ± 0,52
-132,00 ± 1,60
-0,59 ± 0,09
-2,17 ± 0,32
0,987
I3Me4
5,1
52,5
-45,24 ± 0,55
-139,00 ± 1,67
-2,13 ± 0,31
-3,80 ± 0,56
0,990
I3Me4
5,4
44,3
-38,36 ± 0,44
-120,12 ± 1,39
-1,11 ± 0,16
-2,55 ± 0,37
0,993
I3Br2
5,1
38,8
-28,73 ± 0,19
-91,42 ± 0,61
-0,38 ± 0,05
-1,47 ± 0,19
0,995
I3Br2
5,4
31,7
-33,10 ± 0,22
-108,42 ± 0,73
0,53 ± 0,07
-0,76 ± 0,10
0,996
I3Br4
5,1
29,7
-30,86 ± 0,30
-101,65 ± 1,09
0,67 ± 0,09
- 0,55 ± 0,08
0,989
I3Br4
5,4
21,8
-29,49 ± 0,47
-99,65 ± 1,58
1,42 ± 0,23
0,22 ± 0,04
0,979
Die Schmelztemperaturen zeigen in allen Fällen eine pH-Abhängigkeit insofern, dass die
Schmelztemperatur für alle Sequenzen bei pH 5,1 höher war als bei pH 5,4. Im Zusammenhang damit
war auch die Gibbsenergie für alle Sequenzen bei pH 5,1 niedriger als bei pH 5,4. Dies hat den Grund,
dass das i-motif am stabilsten ist, wenn es hemiprotoniert vorliegt, das heißt, wenn der pH seinem
pKa ähnelt. Der pKa wird für Cytosin mit ca, 4,8, für 5-Methylcytosin 5,5 (Moser et al. 2009) und für
5-Bromocytosin mit 2,45 angegeben (Kulikowski und Shugar 1979). Allerdings beinhalten die
Strukturen nur einige substituierte Cytosine, deshalb sollte der Effekt nicht so groß sein wie zunächst
angenommen (wie die Messungen bezüglich des pHs schon gezeigt haben). So ist es zu erklären, dass
für I3Me2 und I3Me4 dennoch die Schmelztemperatur bei pH 5,1 größer ist als bei pH 5,4. Die
berechneten Werte ergeben weiterhin, dass in allen Fällen der Enthalpiewert begünstigend für die
Faltung ist (das heißt, ΔH ist negativ), aber von einer ungünstigen Entropie (das heißt, ΔS ist ebenfalls
negativ) kompensiert wird. Das kommt dadurch zustande, dass die Faltung des i-motifs zwar Energie
freisetzt durch das Bilden der Wasserstoffbrücken und von stacking-Wechselwirkungen zwischen den
Basen, gleichzeitig aber die Freiheitsgrade des Phosphatrückgrads (v.a.) drastisch eingeschränkt
werden (Searle und Williams 1993). Wie schon bei den Betrachtungen der pH-Abhängigkeit, ist der
Effekt der zusätzlichen Cytosine größer als der der Substitution von Cytosin durch 5-Methyl- bzw. 5Bromocytosin. Die zusätzlichen Cytosine und die Methylierungen haben auch hier wieder einen
73
— Zusammenfassung —
stabilisierenden Effekt, die Bromierungen einen destabilisierenden. Für eine genauere Beschreibung
sei auf Kapitel 4.2 verwiesen.
Zusammenfassend und abschließend ist zu sagen, dass die Betrachtung der Sequenzen, die
Substitutionen enthielten, nicht so ergiebig im Hinblick auf die Verwendung für i-motif basierte
Nanodevices war wie erhofft. Die Effekte der Verlängerung der C-reichen Abschnitte waren
deutlicher, aber hier konnte die pH-Abhängigkeit der Faltung bzw. der Faltungsmechanismus nicht
geklärt werden. Die nächsten Schritte wären es also, diese Faltungsmechanismen näher zu
beleuchten. Anschließend könnte man zum Beispiel untersuchen, ob sich die Effekte, die sich mit den
verschiedenen Sequenzveränderungen erzielen ließen, kombinieren lassen, um so ein i-motif zu
erhalten, das unter mehr physiologischen Bedingungen stabil ist.
74
— Reference —
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