Tuning the pH Response of i-Motif DNA OligonucleotidesLaurie Lannes,[a] Saheli Halder,[b] Yamuna Krishnan,[b, c] and Harald Schwalbe*[a]

Introduction

In addition to the well-known double helix conformation, spe-

cific DNA sequences can form additional, more complex ter-tiary structures stabilised by non-Watson–Crick base pairs.

Under specific conditions, cytosine- and guanine-rich sequen-

ces exhibit a rich polymorphism and can form quadruplex sec-ondary structures known as i-motifs and G-quadruplexes.[1]

The i-motif structure consists of a tetraplex composed oftwo anti-parallel duplexes connected by intercalated hemipro-

tonated cytidine·cytidine+ base pairs (C·C+).[1a] This pH-depen-dent protonation of opposite cytidine base pairs can occur

under mild acidic conditions at the N3 position; the pKa of iso-

lated cytosine is 4.58.[2] As a consequence, i-motif sequencesare fully folded within a pH range of 5–6.

The complementary strand can form G-quadruplexes thatare composed of four strands connected by planar G-tetrads

stacked on top of each other. Formation of G-tetrads relies onHoogsteen hydrogen bonds and is often dependent on the

presence of monovalent cations (Na+ , K+ , NH4+), occupying

the central channel between tetrads.[1b]

G-quadruplexes and i-motifs are present in tandem on com-

plementary strands in particular locations of the genome, in-cluding telomeres,[3] oncogene promoters,[4] and centromeres.[5]

The colocalisation of these sequences has generated consider-able interest in understanding their functions and whether

their functions might even be coupled.[6]

Direct evidence of the in vivo existence of i-motifs is still

missing. At first glance, the pH in the environment of the nu-cleus should be too high for i-motifs to form. However, studies

showed that C-rich sequences can form at physiological pH in

a crowded environment[7] or from a duplex under negative su-perhelicity pressure.[8]

Several proteins have been identified to bind i-motif-compe-tent sequences.[9] For example, Hurley et al. recently discovered

the first protein (hnRNP L-like) that recognises and preferential-ly binds to the i-motif conformation over random coil confor-

mations of bcl-2 C-rich promoter sequences (Py39wt).[9e] In ad-

dition, by using ligands that have antagonist effects on i-motifstability and subsequent binding to hnRNP L-like, they were

able to control bcl-2 expression in vitro.[9e, 10]

In addition to their biological functions, the use of DNAs as

building blocks for nanodevices has become an attractive fieldof research. In this field, C-rich DNAs have obtained considera-ble attention due to their unique pH-switching capacity. In

2003, the Balasubramanian group designed the first i-motif-based nanodevice by functionalising it at the 5’ and 3’ terminiwith a fluorophore and a quencher, respectively. A switch inpH allowed cyclic reversible generation of either an i-motif

(low pH) or a duplex (neutral pH).[11] Protonation-dependenttransitions from duplex or random coil conformations to the i-

motif structure have since been implemented to design severalnanodevices. Applications are broad and as various as pH sen-sors,[12] logic gates,[13] electronic components,[14] nanopores for

substrate delivery,[15] or ion nanochannels.[16]

Cellular pH sensors are particularly interesting nanoma-

chines, as the intracellular pH (pHi) has an important role in cel-lular homeostasis. Cells do not maintain identical pH values

throughout, but each compartment has an optimum pH. For

instance, the nucleus and the cytosol have a pH of 7.2, where-as mitochondria adopt a pH of 8.0, the Golgi a pH of 6.0–6.7

and the lysosomes a pH of 4.7.[17] Acidification of the cell, forexample, is linked to apoptosis.[18] Cancer cells undergo basifi-

cation (pHi>pH 7.4), which leads to a reversed pH gradientbetween the intra- and extracellular environments.[18] Hence,

Cytosine-rich single-stranded DNA oligonucleotides are able to

adopt an i-motif conformation, a four-stranded structure, near

a pH of 6. This unique pH-dependent conformational switch isreversible and hence can be controlled by changing the pH.

Here, we show that the pH response range of the human telo-meric i-motif can be shifted towards more basic pH values by

introducing 5-methylcytidines (5-MeC) and towards more

acidic pH values by introducing 5-bromocytidines (5-BrC). No

thermal destabilisation was observed in these chemically modi-

fied i-motif sequences. The time required to attain the newconformation in response to sudden pH changes was slow for

all investigated sequences but was found to be ten timesfaster in the 5-BrC derivative of the i-motif.

[a] L. Lannes, Prof. Dr. H. SchwalbeInstitute for Organic Chemistry and Chemical BiologyCenter for Biomolecular Magnetic Resonance (BMRZ)Johann Wolfgang Goethe-University FrankfurtMax-von-Laue-Strasse 7, 60438 Frankfurt/Main (Germany)E-mail : schwalbe@nmr.uni-frankfurt.de

[b] S. Halder, Dr. Y. KrishnanNational Centre for Biological Sciences, TIFRGKVK Campus, Bellary Road, Bangalore 560065 (India)

[c] Dr. Y. KrishnanDepartment of Chemistry, University of ChicagoE305, GCIS, 929 E, 57th Street, Chicago, IL 60637 (USA)

Supporting information for this article is available on the WWW underhttp ://dx.doi.org/10.1002/cbic.201500182.

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Full PapersDOI: 10.1002/cbic.201500182

monitoring of the pHi is of high interest for diagnostics, drugdesign, and better understanding of cellular processes.

In the case of i-motif-based pH sensors for in vivo applica-tions, several issues drive the design of such switchable DNA

sequences. The device should respond in an adequate pHrange, according to the targeted cellular compartment. When

the organelle of interest undergoes a rapid change in pH, asoccurs in the endosome, the Golgi, or any organelle under pH

stress conditions, the pH sensor should also process a fast re-

sponse in order not to miss monitoring spatial and temporalpH changes. Therefore, it is mandatory to investigate the pH

profile of various i-motifs in terms of the midpoint of titrationand the transition width of the titration, as well as the kinetics

of their folding.In previous work, we characterised the pH-induced folding

pathway of the human telomeric i-motif DNA d[(CCCTAA)3CCC]

by static and time-resolved NMR spectroscopy.[19] Our investi-gations revealed a kinetic partitioning mechanism with a first

step in which two conformations (Scheme 1) are formed with

a rate constant on the order of 2 min¢1. Subsequent refoldingof the kinetically favoured conformation to the thermodynami-cally more stable conformation was slow, with rate constantson the order of 10¢3 min¢1. At equilibrium, two distinct confor-mations were populated at a ratio of 3:1. Cytosine-selectiveisotope labelling schemes allowed us to assign both conform-

ers, which differ in the intercalation topology of the C·C+ basepairs.[19–20] The major conformer is closed by the C·C+ base pairat the 5’-end position (5’E), whereas the minor conformer is

closed by the C·C+ base pair at the 3’-end position (3’E).The human telomeric sequence was previously integrated

into a nanostructure to quantitatively assay the stability andlifetime of various DNA nanostructures in vivo.[21] The mutant

sequence I4, which presents an extra cytosine in each C-tract,

was implemented in an i-motif switch designed to probe thepH evolution of endosomes in real time.[12d]

In this report, we investigated whether the pH response ofthe human telomeric i-motif (I3) can be tuned by substituting

cytosines with 5-methylcytosine (5-MeC) and 5-bromocytosine(5-BrC) or by elongating it with an additional cytosine (I4). This

approach is motivated by the different pKa(N3) values of free5-MeC, 5-BrC, and C. Karino et al. determined that 5-MeC has

a pKa(N3) of 4.5, whereas C has a pKa(N3) of 4.4.[22] Kulikowskiet al. found that 5-BrC has a pKa(N3) at 2.45, compared to 4.1

for C.[23] Further, we investigated the influence of such modifi-cations on the kinetics of i-motif formation at different pH

values.Using various cytosine derivatives and extending the length

of the C·C+ strands allowed us to tune the pH range by ++0.14and ¢0.22 pH units and the folding kinetics by a factor of 10,whereas the previously observed partitioning of the foldingpathways remained unaltered.

Results and Discussion

We rationalised the positions of 5-MeCs and 5-BrCs accordingto the structural organisation of the I3 i-motif.[19, 24] We decided

to position the modified cytosines in order to form homoge-nous base pairing (i.e. , 5-xC·5-xC+ , where x can be a methyl

group, a bromine substituent, or a hydrogen atom). Indeed,

we showed in previous work that the C·C+ imino proton isdynamically bound to both cytidines across the strands, and

hydrogen bonding needs to be described by a double well po-tential, which requires the pKa to be tuned on both sides of

the base pairing.[25]

Furthermore, we introduced predicted chemically modified

C·C+ base pairs in the middle of the C·C+ core, where the

modifications should lead to minimal interactions with loopnucleotides.[19] Scheme 1 presents the i-motif organisation of

the DNA sequences reported in Table 1. Further, C-rich oligonu-cleotides presenting four Cn tracts (with n�2) are also expect-

ed to form an intramolecular i-motif.[6a, 26]

i-Motif folding competence

In order to determine the stoichiometry of the i-motifs after

acidification, we carried out polyacrylamide gel electrophoresis(PAGE). On denaturing PAGE, I3, I3Me4, and I3Br2 migrated inan identical manner compared to a polydT sequence of identi-cal number of nucleotides (polydT T21) (Figure 1 A). Thus, intro-duction of bromo- or methyl-substituted cytidines into the oli-

gonucleotides did not lead to any significant migration differ-ence when DNA molecules were fully relaxed. As a conse-

quence, differences in migration on native PAGE can be inter-

preted as arising from differences in secondary structure. ThepolydT sequences (dT10, dT21, and dT25) were used as size mark-

ers, assuming that their migration behaviour was not affectedby differences in pH. At pH 5.0 (Figure 1 B), the sequences of

interest formed species that migrated roughly together withT10, appearing twice as small as predicted from their actual

Scheme 1. Organisation of the 3’E and 5’E conformers of the human telo-meric i-motif I3 (left) and its mutant I4 (right).[19] The hemiprotonated cyto-sine·cytosine+ (C·C+) base pairs are depicted as full triangles. C2·C14+ andC8·C20+ are composed of 5-methylcytosines, and C8·C20+ is composed of5-bromocytosines in I3Me4 and I3Br2, respectively. Table 1. Oligonucleotide sequences.

Name Sequence 5’!3’ Name Sequence 5’!3’

I3 (CCCTAA)3CCC I4 (CCCCTAA)3CCCCI3Me4 (C5mCCTAA)3C5mCC I3Br2 (C3TA2C5BrCCTA2C3TA2C5BrCC

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size. The intramolecular i-motif structure is more compact than

an ssDNA coil and is expected to migrate faster than dT21 se-

quences without structure. All sequences showed one strongband, indicating that they formed a monomeric structure at

acidic pH. Interestingly, I3Br2 migrated slightly slower than I3and I3Me4, possibly due to formation of a less compact struc-

ture. In both gels, the I3Br2 lane showed a higher light bandthat might correspond to a stable dimer. Certain nucleic acid

secondary structures are not fully disrupted in regular denatur-

ing gel.[27]

We further tested i-motif formation by NMR spectroscopy.

The imino proton engaged in the C·C+ base pair has a charac-teristic chemical shift around 15.5 ppm. Figure 2 shows the 1H

1D spectra of each DNA sequence of interest at acidic pH, fo-cusing on the 16–15 ppm region. In the corresponding NOESYspectra, cross peaks could be observed between intercalated

C·C+ base pairs protons, caused by their close proximity (3.3 æaverage distance, as determined by NMR structure 1EL2;[24] Fig-

ure S1 in the Supporting Information). This cross-peak patternprovided additional evidence for i-motif formation. Notably,

the I3Br2 1D spectrum presented minor peaks around 14.5–15 ppm that could belong to C·C+ imino protons from the

putative dimer form already observed by electrophoresis.[28]

We assessed the proportion of this species as ~5 % at NMRconcentration. This marginal population was considered not

important in following experiments.

pH and thermal stability

We monitored pH-dependent i-motif formation by circular di-

chroism (CD) spectroscopy. The resulting CD spectra acquiredover the pH range 7.2 to 4.8 are presented in Figure 3. The

I3Me4 and I3Br2 sequences revealed similar spectral character-istics to I3 and I4. At pH 5.0, the oligonucleotides (ODNs) dis-

played a maximum band around 288 nm and a minimum bandbetween 255 and 260 nm (individual values are given in

Table 2), in agreement with previous reports.[4b, d, g, 29] At pH 7.2,the ODNs had a complete different profile, with a maximum

band near 275 nm and a minimum band near 250 nm, charac-teristic of a single-stranded DNA random coil conformation.[30]

In addition, pH titration of the CD spectra of I3, I4, and I3Br2revealed two distinct isoelliptic points, which represent strong

evidence for a transition between two discrete conformational

states.[31] The pH-dependent CD spectra showed that the non-natural nucleotides did not impair the formation of i-motif

structure. The introduction of 5-MeCs into i-motif sequenceshas already been studied, and similar results as reported

herein have been observed.[24, 32] On the other hand, the intro-duction of 5-BrCs was never reported.

Figure 1. A) 20 % denaturing (8 m urea) polyacrylamide gel (PAGE). ThepolydT dT10, dT21, and dT25 were size marker oligonucleotides. B) 20 % nativePAGE, buffered by TAE pH 5.0. Bands were visualised by UV shadowing.

Figure 2. A) Hemiprotonated cytidine·cytidine+ (C·C+) base pairs witha proton shared by both cytidines, as described by Lieblein et al. in 2012.[25]

B) C·C+ base pair imino proton region of 1D NMR spectra of i-motif DNAsequences I3, I4, I3Me4, and I3Br2 at slightly acidic pH.

Table 2. Characteristics of the CD spectra of i-motif DNA sequences.

I3 I4 I3Me4 I3Br2

max. band[a] [nm] 288.3 287.7 288.3 288.5(275.4) (275.7) (274.5) (275.2)

min. band[b] [nm] 257.3 260.3 254.7 254.5(248.3) (247.1) (249.8) (248.3)

isoelliptic points 277.0 278.0 276.6 278.0[�0.2 nm] 246.0 243.2 n.o. 244.2

[a] Average of values obtained in triplicate for 100 % fraction folded/un-folded. [b] Average of spectra measured in triplicate. n.o. : not observed.Band values in brackets correspond to the unfolded state.

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We chose the molar ellipticity at 288 nm for I3, I3Me4, and

I4 and 289 nm for I3Br2 as a reporter (ME288/289) for i-motif for-

mation to follow the state of folding for each of the sequencesover a pH range between 4.8 and 7.2. The pH-dependent fold-

ing was fully cooperative for all systems investigated. At thelowest and highest pH values, we observed maximal and mini-

mal ME288/289, respectively. We conclude that the pH-inducedtransition can be well titrated over the chosen pH range. Con-

sequently, we converted the ME288/289 into the DNA fraction

folded (FF). Plots of the FF against pH values are presented inFigure 4.

In order to use an i-motif as a pH sensor, the midpoint ofthe pH-dependent cooperative folding/unfolding transition

must coincide with the (cellular) pH of interest. We defined thewidth of the pH transition as the pH response range of the C-

rich sequence. In order to compare the pH response ranges of

the various i-motifs studied, we defined this within an upperand a lower pH limit as defined by 95% and 5 % of the fraction

folded (FF95 and FF5, respectively; Table 3).Considering the transitional pH (pHFF50) of each sequence,

we noticed that 5-BrC had the opposite effect of 5-MeC.Indeed, I3Br2 revealed a DpHFF50 (pHFF50 (I3)–pHFF50 (I3Br2)) of

¢0.33, whereas I3Me4 had a DpHFF50 of ++0.14. In comparison,the elongation of the C-tracts in I4 showed a larger DpHFF50 for++0.38 than for I3Me4. Further, by analysing the amplitude of

the pH response range of the sequences, differences in the co-

operativity of folding were ap-parent. The transition range

(pHFF95–pHFF5) of I3 spanned0.69 units. The pH response

range of I3Br2 was narrower,with a transition range of 0.54,

contrary to I3Me4, whichshowed a broader transition of

0.85. As a result, the introduction

of 5-MeCs led to a decrease incooperativity of the pH-induced

folding transitions, contrary towhat was observed upon intro-

duction of 5-BrCs.From PAGE analysis, we dem-

onstrated that the investigated

sequences adopted an intramo-lecular, monomeric i-motif. Onthis basis, we calculated the

thermodynamic parameters from CD temperature denaturation

curves. We measured melting curves at two different pHvalues. Here, it is relevant to compare the thermodynamic pa-

rameters of the sequences at a pH value within their pH re-sponse region, leading to the same fraction of folded i-motif.

Consequently, we chose to measure melting curves at pHFF50.In addition, we measured melting curves at pH 5.0. The result-

ing melting curves are presented in Figure 5.

As expected, the melting temperature (Tm) of each sequencedecreased as the pH increased (see Table 4). Based on the Tm

values at pH 5.0, I3Me4 was the most stable i-motif comparedto I3 and I3Br2, with the latter being the least stable. Interest-

ingly, at their respective pHFF50 values, these sequences hadsimilar Tm values.

Figure 3. CD spectra of i-motif-competent sequences I3, I3Me4, I3Br2, and I4 over the pH range 4.8–7.2. Thepresented spectra were averaged over three successive acquisitions. The ellipticity was converted into molarellipticity.

Figure 4. pH melting curves of i-motif sequences I3, I3Me4, I3Br2, and I4over the pH range 4.8–7.2 at 298 K. The plots are derived from molar elliptic-ity at 288 nm (I3, I4, and I3Me4) or 289 nm (I3Br2), monitored during the pHtitration presented in Figure 3. The CD data were transformed into foldedfraction and plotted against pH values. Fitting was performed by using fivepoints measured in triplicate. The error bar dots were obtained by averagingthe values of the triplicate measures, the limits of the error bars correspondto the highest and the lowest values.

Table 3. pH-response range of i-motif sequences.

95 % folded 50 % folded 5 % folded

I3 5.90 6.26�0.01 6.59I3Me4 5.88 6.40�0.02 6.73I3Br2 5.68 5.93�0.01 6.22I4 6.32 6.64�0.01 6.91

� standard error from fitting in Figure 4.

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At pH 5.0, all sequences were folded both at 25 and 37 8Cexcept I3Br2, of which 20 % were in random coil DNA struc-

tures at 37 8C. At pHFF50, we observed reduced stability. Indeed,at 25 8C, the i-motif populations of I3, I3Me4, and I3Br2 de-creased by a third and almost completely disappeared at 37 8C.

I4 presented a higher stability, due to its extra two C·C+ basepairs. Consequently, considering its pHFF50, the I4 i-motif at

25 8C was largely formed, but its folded conformation de-creased by two-thirds at 37 8C. Nevertheless, for I4, the quadru-

plex form was still populated at its pHFF50, contrary to theother sequences studied.

Recently, Xu et al. investigated the introduction of one ortwo 5-MeCs at different positions into the I3 sequence. Theyreported that the position of one modified cytosine showeda limited influence on pHFF50. However, they observed a greatereffect on the pH response range when both cytosines of

a C·C+ base pair were substituted instead of one. This observa-tion confirmed the relevance of our tuning approach discussed

above.[32c] Interestingly, Bhavsar-Jog et al. reported a DpHFF50 of

++0.2 when the cytidine at position 4 in d[TTC3TAC4AC3TA2]ODN was replaced by a 5-MeC, due to a decrease in coopera-

tivity.[33] The introduction of only one 5-MeC into I3 leads toa more modest DpHFF50,[32c] and it is possible that different

parental sequences then undergo different effects due to themodified cytidine introduction. In the case of I3Br2, it was strik-

ing that the exchange of only one C·C+ base pair by a homo5-BrC·5-BrC+ base pair led to such tremendous effect on the I3

pH response range. The introduction of one 5-hydroxymethyl-cytosine (5-hmC) in i-motif sequences also led to an acidic tu-

ning.[32c, 33]

Yang et al. measured the base-pairing energies (BPEs) of 5-xC·5-xC+ homodimers by guided ion beam tandem mass spec-troscopy. They revealed that 5-MeC·5-MeC+ dimers compriseda higher BPE than C·C+ dimers (177.4 and 169.9 kJ mol¢1, re-spectively).[34] In line with these findings, we propose that 5-MeC·5-MeC+ base pairs in i-motifs are more stable than C·C+

base pairs. As a result, the pH response range might be broad-ened. Due to its lower pKa(N3), 5-BrC has a weaker proton

affinity than C. As a consequence, the 5-BrC·5-BrC+ base pairmight disrupt at more acidic pH than do C·C+ base pairs. How-

ever, 5-BrC·5-BrC+ dimers show only a slightly lower BPE

(168.5 kJ mol¢1) than the unmodified parent, which impliesthat both base pairs have similar stability.[34]

Kinetic investigations

In previous work, we elucidated the folding kinetics pathway

of the human telomeric sequence I3. We established that1) the folding proceeds in two steps, and 2) two different

folded i-motif conformations are present at equilibrium. Initial-ly, the less stable conformation is formed more rapidly, but at

equilibrium, two conformations are present: 5’E is three times

more populated than the 3’E conformer.[19]

Here, we first investigated whether this complex folding

pathway was conserved in the investigated i-motif sequences.Thus, we performed time-resolved NMR spectroscopy to follow

the folding of the I4 sequence and used the NMR characteristicchemical shift around 15.5 ppm arising from the proton shared

in the C·C+ base pairs.

After initiating folding by a temperature jump from 95 to25 8C, we followed the evolution of successive 1D 1H NMR

spectra over a period of 24 h. Figure 6 A shows a sample ofspectra, focusing on the imino proton region, at different timepoints. Eight peaks would be expected for each conformer;however, due to overlap, only six apparent peaks were clearly

observed. We determined the intensity of each peak. Thesedata were then plotted against time to obtain the kinetic

traces presented in Figure 6 B. Given the large effort requiredto assign individual resonances for the two-state population ofi-motifs,[19–20, 25, 35] we decided to focus here on analysis of the

kinetics without individual assignment, because the kineticprocess did not reveal any differences between different nucle-

otides in the sequence; in other words, there were no singlenucleotide-specific variations.

The four strongest peaks (at 15.77, 15.56, 15.48, and

15.41 ppm) showed a constant increase before reaching a pla-teau. The peaks at 15.34 and 15.53 ppm showed an increase

during the first 30 min before decrease and finally reacheda plateau. The number of states involved in the kinetics of fold-

ing therefore remained unchanged compared to our previousstudies of the I3 sequence, allowing us to conclude that the

Figure 5. CD temperature melting curves of i-motif sequences I3, I3Me4,I3Br2, and I4 at pH 5.0 (*) and at their transitional pH values (*) of 6.3 (I3),6.6 (I4), 6.4 (I3Me4), and 5.9 (I3Br2). The molar ellipticities at 288 nm (I3,I3Me4, I4Br2, and I4) or at 289 nm (I3Br2) were monitored and normalised tobe expressed as fraction folded.

Table 4. Melting temperatures (Tm) of the i-motif sequences at differentpH values, pH 5.0, and transitional pH values.

pH Tm[a] [8C] Tm

[b] [C] pH Tm[a] [8C] Tm

[b] [C]

I3 5.0 55.7 54.8�0.1 6.3 26.4 25.8�0.1I3Me4 5.0 59.1 57.0�0.2 6.4 27.5 27.6�0.1I3Br2 5.0 45.5 44.6�0.1 5.9 26.4 26.6�0.1I4 5.0 68.4 67.9�0.2 6.6 35.1 34.1�0.1

[a] Median line method. [b] From fitting to f = y0 + a/1 + exp((¢x¢x0)/b), �standard error.

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kinetics of I4 folding followed the same model as I3 (see Fig-

ure 6 C).We then studied folding of all sequences by CD spectrosco-

py to investigate folding kinetics in more detail. Depending on

the kinetics, i-motif folding was initiated by a pH jump usingan automatic mixing device (stopped-flow system) or bymanual mixing. We titrated folding kinetics over the pH ramp:pHFF100, pHFF75, pHFF50, and pHFF25. Similar to the melting curvesdescribed above, the characteristic i-motif CD signature at288 nm was monitored over time after the pH jump. After

baseline and zero corrections, the molar ellipticity at 288 nmwas plotted against time to obtain the kinetic traces presentedin Figure S3. Most of the collected data could be fitted bysingle or double exponential functions. Therefore, F-tests weresystematically run to compare the two possible fits for model

selection. According to these statistical tests, the best fittingwas always obtained by using a double exponential function.

The two rate constants describing the complex folding path-way are given in Figure 7 and Table S5.

It is striking how the pH influences the folding kinetics of I3,

I3Me4, and I4. For these sequences, folding was greatly decel-erated when we compared a protonation change at saturating

pH (~pH 8 to 5) to a change at non-saturating pH (~pH 8 topHFF75, for example), as previously observed for I3.[36] In fact, it

took 0.5, 10, and 4 s for I3, I3Me4, and I4, respectively, to reachthe equilibrium plateau after a pH jump from 8 to 5, whereasit took more than 1000 s after a smaller amplitude pH jump.

Interestingly, only I3Br2 did not show such strong effects. ForI3Br2 folding, equilibrium was reached in 100 s for a pH jumptowards saturating value; however, it took 150 and 200 s when

jumping to non-saturating pH values.The folding rate constants k1 and k2 in Figure 7 reflected

these observations. When we compared the rate constants ob-tained for the saturating pH jump and the non-saturating pH

jumps, we observed differences of a factor of 1000, 100, and10 000 for I3, I3Me4, and I4, respectively. It is noteworthy topoint out that both rate constants were affected in a similarmanner for the same pH jump. In contrast, rate constantsobtained at non-saturating pH jumps revealed no significant

differences.Surprisingly, Chen et al. , who studied pH-induced folding of

I3 by stopped-flow CD spectroscopy, reported that the singleexponential function was the best to describe their data.[36] Ac-cording to our observations, CD spectroscopy is a reliable

method to monitor the complexity of the i-motif folding mech-anism, and all CD kinetic traces had to be fitted to a double

exponential function. Although CD spectroscopy was unableto detect the different conformers 3’E and 5’E determined by

Figure 6. I4 folding kinetics investigation by time-resolved NMR. A) C·C+ base pairs imino proton region of NMR spectra recorded over 24 h. B) Imino protonpeaks intensities are plotted as a function of time to give kinetic traces. The grey dots correspond to the experimental data, and the orange dots correspondto double exponential fitting f(t) = a Õ (1¢exp(¢k1 Õ t)) + c Õ (1¢exp(¢k2 Õ t)). An arrow in the inserted spectra highlights the peak analysed. C) Model of the I4folding pathway from the unfolded state (U) towards i-motif structures partitioned between two conformers, according to the model published by Liebleinet al.[19]

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NMR spectroscopy, including single-labelled nucleotides,[19] be-

cause their optical signatures are equivalent, the partitioningof the folding pathway between the two conformers can nev-

ertheless be determined unambiguously and characterised bytwo constant rates.

According to our data for I3, I3Me4, and I4, both foldingsteps were influenced by the proton concentration or, more

precisely, by a pH range, described as protonation-saturatingand -non-saturating. Only the I3Br2 case was different becauseonly the first rate constant (k1) showed this behaviour. It ap-peared clear that the first conversion from a random coil DNA

conformation to the i-motif, with preferred formation of theless stable 3’E conformer, revealed a pH dependence. However,the second step involved a conversion from the 3’E conformer

to 5’E conformer. The observation of a pH dependence for thissecond step, which involves structural rearrangement between

two folded conformations, suggests that C·C+ base pairs needto be deprotonated to be disrupted, and reprotonation is re-

quired for the formation of the compact i-motif. This deproto-nation/reprotonation step, in turn, leads us to propose that un-

folded or partially unfolded intermediates need to be involved

in structural conversion. Interestingly, I3 and I4 showed similark1 rate constants, but the k2 constant rate for I4 was tenfold

smaller than for I3. This finding suggested that only thesecond step, but not the first folding step, was affected by the

number of C·C+ base pairs to be formed. This finding suggeststhat formation of the base pairs in the first step is simultane-

ous, and that conversion of the two conformers is influenced

by the extra two C·C+ base pairs.

Conclusion

Because of the slightly higher and significantly lower pKa

values of the N3 atoms of 5-MeC and 5-BrC, respectively, we

were able to tune the pH response of i-motif DNA oligonucleo-tides. NMR and CD spectroscopy showed that the chemical

modifications do not prevent the studied DNAs from formingan i-motif at slightly acidic pH values. Gel electrophoresis re-

ported the formation of only intramolecular folding. The new

sequences containing 5-MeCs and 5-BrCs displayed a coopera-tive pH response. This behaviour makes them suitable for im-

plementation in nanodevices. Introduction of 5-MeCs in I3Me4decreases the cooperativity of folding and therefore broadens

the pH response range, especially toward more basic values,which could make I3Me4 suitable for monitoring the Golgi net-work pH between 6 and 6.7 (I3Me4 responds over a pH rangeof 5.88–6.73).[17] Elongation of the C-tracts is also an interesting

strategy, which was already explored,[12a] to tune the responsetoward more basic values. I4 can monitor a more basic pHthan I3Me4, but once the pH response range is shifted, then

the acidic range detectable by I3 is lost with I4. On the contra-ry, the introduction of 5-BrCs in I3Br2 leads to the opposite

effect: the pH response range is shifted towards more acidicvalues.

We did not observe thermal destabilising effects, due to the

introduction of modified cytosine residues. Nevertheless, wefound out that I3, I3Me4, and I3Br2 i-motifs are poorly populat-

ed at 37 8C at their respective transitional pH values, whichmakes them suboptimal for applications at physiological tem-

perature. Thus, I4 sequences present an advantage, becausethey still show a large i-motif population at 37 8C.

Figure 7. Folding rate constants k1 and k2 of I3, I3Me4, I3Br2, and I4, corre-sponding to different pH jumps. The folding of the DNA sequences was trig-gered by a pH jump from pH 8 to acidic pH values by using stopped-flowmixing or manual mixing. The folding was then monitored by circular di-chroism at 288 nm. The kinetics were titrated over a pH ramp composed ofone protonation-saturating pH jump (pH 8 to 4.89 or 4.96) and three proto-nation-non-saturating pH jumps. The rate constants k1 and k2 were obtainedby fitting the kinetic traces of Figure S3 to a double exponential function.The average of the k2 values for the non-saturating pH jumps of I3, I4, andI3Me4 and the average of all k2 values of I3Br2 were plotted; error bars cor-respond to the maximum and the minimum values. *Standard error from fit-ting. **Maximum and minimum k2 values.

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Finally, we demonstrated that the partitioned folding path-way of the i-motif is conserved for all studied C-rich sequences.

The introduction of 5-MeC does not change the folding kineticrate, compared to I3. On the contrary, 5-BrC accelerates the

folding kinetics. Elongation of the C-tracts leads to a decreasein the folding rate, due to a much slower conformer conver-

sion during the second step of the folding mechanism. As aconsequence, I4 needs hours to reach equilibrium.

I3 analogue I3Me4 showed that the introduction of 5-MeC

slightly decreased the cooperativity of folding and broadenedthe pH response range toward more basic values (++0.19). We

could imagine that the introduction of 5-MeCs in I4 would pro-duce a similar effect. Knowing that I4 has a pH response range

between 6.3 and 6.9, the addition of 5-MeCs might lead to thedesign of DNA sequences that could monitor neutral and

slightly basic pH conditions. Similarly, we could transfer to 5-

BrC-I4 analogues the properties observed for I3Br2. Weshowed that 5-BrC introduction shifted the pH response range

of the parental sequence towards more acidic values and ac-celerated the folding kinetics. The resulting pH response range

of a 5-BrC-I4 analogue might overlay the I3-responsive pHrange. This I4 analogue would have an advantage over I3 in

that it would have thermal stability. In addition, we showed

that 5-BrCs could accelerate I4 folding kinetics.The tuning strategies present advantages and disadvantages

on different levels which inevitably require compromises if ap-plied to nanodevices. The comprehensive biophysical analysis

presented here shows that it is paramount to perform a com-plete analysis with thermal stability and kinetic investigations

of i-motif sequences for optimisation in their application as cel-

lular nanodevices.We decided to target C·C+ base pairs in our tuning tactics,

but the analysis of i-motif sequences found in promotor se-quences, as in bcl-2 or c-myc, suggests that long loops induce

a pH stabilisation effect.[29] The loops are suspected to havea capping effect. The design of the loops in order to optimisethe formation of stacking of loop nucleotides with a C·C+-cyti-

dine core, by using Watson–Crick (WC) or non-WC base pairinginto the loops, represents an interesting direction to follow.The 5’- and 3’-end regions could also be exploited to introducesupplementary stabilising elements. For instance, Nesterova

et al. shifted the transitional pH of the ODN d[(C5T3)3C5] from6.9 to 7.2 by introducing a C-rich sequence in the loop of

a hairpin, with the stem of the hairpin leading to the stabilisa-tion of the i-motif with regard to pH and temperature.[37]

In summary, the biophysical optimisation of the pH response

for various i-motifs, using natural and non-natural cytosine de-rivatives with regard to stability and folding kinetics, will pro-

vide key insights for applications in bio-nanotechnology andbeyond for optimising sequence–response relationships for

this exciting class of tuneable oligonucleotides.

Experimental Section

DNA oligonucleotides: Oligonucleotides were purchased from Eu-rofins MWG Operon. After HPLC purification, DNA samples werefreeze-dried and desalted by using microconcentrators with

a 3 kDa cut-off (Vivaspin 2, Sartorius). For the I4 ODN, a LiClO4/ace-tone precipitation was performed to replace DNA counterionsfrom the HPLC step with lithium, and the ODN was then dissolvedin water. DNA concentrations were determined by UV/Vis spectros-copy on a Cary50 UV-spectrophotometer by using extinction coeffi-cients at 260 nm (e260), as presented in Table 5.

Sample preparation: Samples for circular dichroism (CD) spectros-copy experiments had DNA concentrations of 19.5–20 mm for I3, I4,I3Me4, and I3Br2, and all samples were prepared from the samestock solution. The oligonucleotides were buffered by 25 mm po-tassium acetate buffer over the pH range 4.8–5.6, or by 25 mm po-tassium phosphate buffer over a pH range of 5.8–7.2. The sampleswere incubated at 95 8C for 10 min and left at 4 8C at least one dayfor equilibration before measurement.

Samples for static NMR spectroscopy were systematically snap-cooled before measurement. DNA concentrations were 2 mm (I3)600 mm (I4), 150 mm (I3Br2), and 70 mm (I3Me4). The samples werebuffered by 25 mm potassium phosphate buffer at pH 5.3 (I3 andI3Br2) or 5.5 (I4 and I3Me4). The samples were supplemented with10 % of D2O, and DSS was used as an internal reference.

The I4 NMR sample for kinetics measurement was composed of300 mm I4 DNA, 25 mm potassium phosphate buffer (pH 6.4), and10 % D2O; DSS was used as an internal reference. The sample wasincubated 5 min at 95 8C immediately prior to acquisition.

Denaturing PAGE: 20 % polyacrylamide gel was mixed with 1 ÕTBE buffer pH 8.3 and urea (8 m). I3, I4, I3Me4, I3Br2, T10, T21, andT25 (150 mm each) were combined with loading buffer (99 % forma-mide, 0.01 % bromophenol) and loaded on the gel. The gel wasrun at room temperature in 1 Õ TBE buffer with a current of 220 Vapplied. Band migration was revealed on a silica gel plate underUV light shadowing and photographed with a digital camera.

Native PAGE: 20 % polyacrylamide gel was prepared by using 1 ÕTAE buffer, pH 5.0, 4 8C. DNA samples were combined with loadingbuffer (50 % glycerol, 5 Õ TAE buffer, pH 5.0, 4 8C). All samples wereincubated at 95 8C for 10 min and stored overnight at 4 8C beforeloading. The gel was run at 4 8C in 1 Õ TAE buffer with a current of60 V applied. DNA bands were visualised on a silica gel plate underUV light, and the gel was then photographed with a digitalcamera.

CD spectroscopy methods: The CD spectra and temperature melt-ing curves were recorded on a Jasco J-810/815 CD spectropolarim-eter equipped with a Jasco PTC-4235L Peltier thermostated cellholder. The cell chamber was flushed with a constant nitrogenflow to avoid water condensation on the measurement cuvette.

Table 5. Oligonucleotide sequences and their extinction coefficients(e260).

Name Sequence 5’!3’ e260 [m cm¢1]

I3 (CCCTAA)3CCC 185 900[a]

I4 (CCCCTAA)3CCCC 214 700[a]

I3Me4 C5mCCTA2C5mCCTA2C5mCCTA2C5mCC 180 450[b]

I3Br2 C3TA2C5BrCCTA2C3TA2C5BrCC 178 830[b]

[a] Calculated by using the nearest-neighbour model. [b] Calculated byusing the base composition method. For 5-MeC and 5-BrC, e260 values of5.7 and 3.1 mL mmol¢1, respectively, were used.

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For all measurements, a CD quartz cuvette with a path length of1 mm was used.

The CD spectra were recorded at 25 8C over a spectral window of220–330 nm, with data sampling of 0.2 nm at a scan speed of50 nm min¢1. Spectra were the result of the average of three suc-cessive acquisitions. A baseline correction was applied by using anadequate buffer solution. Only the points at critical pH values (thatis, plateaus, inflection points, and transitional points) were mea-sured in triplicate on independent samples to obtain error bars.The ellipticity values were transformed into molar ellipticity, ac-cording to Equation (1):

½q¤ ¼ ðq MÞ=ðc  l   10Þ ð1Þ

where [q] is the molar ellipticity [deg cm2 dmol¢1] , q is the ellipticity[mdeg], M the molecular weight [g mol¢1] , l is the path length[cm], and c is the concentration of the sample in [g mL¢1] . Themolar ellipticity values at 288 nm (I3, I3Me4, and I4) or 289 nm(I3Br2) for each pH point were extracted and normalised to beexpressed as the fraction folded (1 or FF). Data for the triplicatepoints were fitted with a three-parameter sigmoidal function: f = a/(1 + exp(¢(x¢x0)/b)) by using SigmaPlot 12.5 software. x0 corre-sponds to the transitional pH.

Temperature denaturing curves were obtained by monitoring theCD at 288 or 289 nm along a temperature gradient of 4–95 8C, ata rate of 0.5 8C min¢1. One data point was recorded every 0.5 8C.The measured data were normalised and expressed as fractionfolded. We determined the melting temperature by the medianline method[38] and by fitting.

Stopped-flow CD: SFCD measurements were carried out ona Pistar-180 system (Applied Photophysics) set up for CD detection.DNA sample solutions (10 mm DNA in buffer A: 45 mm KCl, 2.5 mmK2PO4, pH 8.02) were rapidly mixed with buffer solution (buffer B:25 mm K2HPO4 at different pH values) in a 1:1 ratio. The mixturecreates a pH jump in the direct environment of DNA moleculesfrom basic to acidic conditions. All acquisitions were performed at25 8C through a path length set at 10 mm. The K+ cation concen-tration was kept constant before and after mixing (50 mm). CDevolution was monitored as function of time at 288 nm witha bandwidth of 8 nm. Kinetics traces were recorded over differentperiods, according to the ODN and the conditions (2–1000 s). Tenthousand points were recorded for each trace, independent of theacquisition time. Each condition was recorded five times (for traces�200 s) or ten times (for traces <200 s) and then averaged. A t0

point was recorded for each condition by mixing DNA solutionagainst buffer A. Baseline corrections (buffer A against buffer A,and buffer A against buffer B) were performed for each conditionand the t0 point, according to the same parameters and repetitionnumber of the corresponding pH jump experiment. The ellipticitywas baseline and zero corrected before being converted intomolar ellipticity (ME), as defined previously. In order to improvethe signal/noise ratio, we averaged the ME of five successive timepoints, (except for I3 kinetics at pHFF75 and pHFF50) and plotted theresult against time.

The kinetics of I4 at pHFF75, pHFF50, and pHFF25 were recorded on thesame “normal” CD spectropolarimeter as describe above. Themixing was manually performed, which led to a dead time of 15 sbefore the beginning of the measure. The temperature was set at25 8C, and the path length was 10 mm. A full spectrum (220–330 nm) was recorded every 2 min with data sampling at 0.5 nm,at a scan speed of 100 nm min¢1, over about 120 min, which repre-

sented 61 spectra in total. The same baselines and t0 point were re-corded as described for stopped-flow CD kinetics measures. Eachkinetics experiment was performed once. After baseline and zerocorrections, the ellipticity was converted into ME. The ME values at287.5, 288.0, and 288.5 nm were averaged and plotted againsttime.

Traces presented in Figure S4 were fitted to a 4-parameter or 5-parameter double exponential function respectively, in SigmaPlot12.5:

MEðtÞ ¼ a  ð1¢expð¢b  tÞÞ þ c  ð1¢expð¢d   tÞ

and

MEðtÞ ¼ ME0 þ a  ð1¢expð¢b  tÞÞ þ c  ð1¢expð¢d   tÞ

An F-test was systematically run to define the best fitting function.

Static NMR spectroscopy: Spectra were recorded on a Bruker600 MHz (I3, I3Br2, and I3Me4) or a 950 MHz (I4) spectrometerequipped with a cryogenic probe at 298 or 288 K (I3). The 1H 1Dspectra used jump-and-return for water suppression,[39] with ajump-and-return delay set at 30 ms (I3), 37 ms (I3Me4), 38 ms (I3Br2),and 15 ms (I4).

Time-resolved NMR spectroscopy: Folding kinetics were moni-tored by real-time NMR spectroscopy on a Bruker 600 MHz spec-trometer equipped with a cryogenic probe at 298 K. The samplewas incubated for 5 min at 95 8C, just before acquisition of apseudo-2D experiment with a jump-and-return sequence for watersuppression.[39] This pulse sequence recorded successive 1D 1Hspectra at several time points. The jump-and-return delay time wasset to 25 ms, the carrier frequency in the proton dimension was setto the water frequency, and the repetition delay was 1 s. A total of16 384 1D spectra were recorded with 5.3 s per spectrum, corre-sponding to an accumulation of four scans. After the five denatur-ing minutes and the first spectral acquisition, 6.5 min elapsed.Kinetic data were processed with TopSpin 3.2 (Bruker Biospin).Kinetics data were fitted to a double exponential function withSigmaPlot 12.5:

Iimino peak ¼ a  ð1¢expð¢b  tÞÞ þ c  ð1¢expð¢d   tÞÞ

where Iimino peaks corresponded to the extracted intensity of iminopeaks, and the coefficients b and d corresponded to two rate con-stants (k1 and k2) describing kinetic partitioning.[19]

Acknowledgements

The authors thank Dr. Boris Fìrtig and Irene Bessi for insightfuldiscussion and Dr. Alexey Cherepanov for stopped-flow CD sup-

port. H.S. is member of the DFG-funded cluster of excellence :

macromolecular complexes. BMRZ is supported by the state ofHessen.

Keywords: bromocytidine · cytosine-rich DNA · i-motifs ·methylcytidine · nanodevices · pH sensors · telomeric DNA

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Manuscript received: April 8, 2015

Accepted article published: May 28, 2015

Final article published: June 30, 2015

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