DOI: 10.1002/cbic.201500182

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Tuning the pH Response of i-Motif DNA Oligonucleotides Laurie Lannes,[a] Saheli Halder,[b] Yamuna Krishnan,[b, c] and Harald Schwalbe*[a] 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 is reversible and hence can be controlled by changing the pH. Here, we show that the pH response range of the human telomeric 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 modified i-motif sequences. The time required to attain the new conformation in response to sudden pH changes was slow for all investigated sequences but was found to be ten times faster in the 5-BrC derivative of the i-motif.

Introduction In addition to the well-known double helix conformation, specific DNA sequences can form additional, more complex tertiary structures stabilised by non-Watson–Crick base pairs. Under specific conditions, cytosine- and guanine-rich sequences exhibit a rich polymorphism and can form quadruplex secondary structures known as i-motifs and G-quadruplexes.[1] The i-motif structure consists of a tetraplex composed of two anti-parallel duplexes connected by intercalated hemiprotonated cytidine·cytidine + base pairs (C·C + ).[1a] This pH-dependent protonation of opposite cytidine base pairs can occur under mild acidic conditions at the N3 position; the pKa of isolated cytosine is 4.58.[2] As a consequence, i-motif sequences are fully folded within a pH range of 5–6. The complementary strand can form G-quadruplexes that are composed of four strands connected by planar G-tetrads stacked on top of each other. Formation of G-tetrads relies on Hoogsteen 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 complementary strands in particular locations of the genome, including telomeres,[3] oncogene promoters,[4] and centromeres.[5] The colocalisation of these sequences has generated considerable interest in understanding their functions and whether their functions might even be coupled.[6] [a] L. Lannes, Prof. Dr. H. Schwalbe Institute for Organic Chemistry and Chemical Biology Center for Biomolecular Magnetic Resonance (BMRZ) Johann Wolfgang Goethe-University Frankfurt Max-von-Laue-Strasse 7, 60438 Frankfurt/Main (Germany) E-mail: [email protected] [b] S. Halder, Dr. Y. Krishnan National Centre for Biological Sciences, TIFR GKVK Campus, Bellary Road, Bangalore 560065 (India) [c] Dr. Y. Krishnan Department of Chemistry, University of Chicago E305, GCIS, 929 E, 57th Street, Chicago, IL 60637 (USA) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201500182.

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Direct evidence of the in vivo existence of i-motifs is still missing. At first glance, the pH in the environment of the nucleus 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 superhelicity pressure.[8] Several proteins have been identified to bind i-motif-competent sequences.[9] For example, Hurley et al. recently discovered the first protein (hnRNP L-like) that recognises and preferentially binds to the i-motif conformation over random coil conformations of bcl-2 C-rich promoter sequences (Py39wt).[9e] In addition, by using ligands that have antagonist effects on i-motif stability 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 field of research. In this field, C-rich DNAs have obtained considerable attention due to their unique pH-switching capacity. In 2003, the Balasubramanian group designed the first i-motifbased nanodevice by functionalising it at the 5’ and 3’ termini with a fluorophore and a quencher, respectively. A switch in pH allowed cyclic reversible generation of either an i-motif (low pH) or a duplex (neutral pH).[11] Protonation-dependent transitions from duplex or random coil conformations to the imotif structure have since been implemented to design several nanodevices. Applications are broad and as various as pH sensors,[12] logic gates,[13] electronic components,[14] nanopores for substrate delivery,[15] or ion nanochannels.[16] Cellular pH sensors are particularly interesting nanomachines, as the intracellular pH (pHi) has an important role in cellular 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, whereas 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, for example, is linked to apoptosis.[18] Cancer cells undergo basification (pHi > pH 7.4), which leads to a reversed pH gradient between the intra- and extracellular environments.[18] Hence,

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Full Papers monitoring of the pHi is of high interest for diagnostics, drug design, and better understanding of cellular processes. In the case of i-motif-based pH sensors for in vivo applications, several issues drive the design of such switchable DNA sequences. The device should respond in an adequate pH range, according to the targeted cellular compartment. When the organelle of interest undergoes a rapid change in pH, as occurs in the endosome, the Golgi, or any organelle under pH stress conditions, the pH sensor should also process a fast response in order not to miss monitoring spatial and temporal pH changes. Therefore, it is mandatory to investigate the pH profile of various i-motifs in terms of the midpoint of titration and 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 investigations revealed a kinetic partitioning mechanism with a first step in which two conformations (Scheme 1) are formed with

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

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Results and Discussion We rationalised the positions of 5-MeCs and 5-BrCs according to the structural organisation of the I3 i-motif.[19, 24] We decided to position the modified cytosines in order to form homogenous 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 is dynamically bound to both cytidines across the strands, and hydrogen bonding needs to be described by a double well potential, 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 loop nucleotides.[19] Scheme 1 presents the i-motif organisation of the DNA sequences reported in Table 1. Further, C-rich oligonucleotides presenting four Cn tracts (with n Š 2) are also expected to form an intramolecular i-motif.[6a, 26]

Table 1. Oligonucleotide sequences.

a rate constant on the order of 2 min¢1. Subsequent refolding of the kinetically favoured conformation to the thermodynamically more stable conformation was slow, with rate constants on the order of 10¢3 min¢1. At equilibrium, two distinct conformations were populated at a ratio of 3:1. Cytosine-selective isotope labelling schemes allowed us to assign both conformers, which differ in the intercalation topology of the C·C + base pairs.[19–20] The major conformer is closed by the C·C + base pair at 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 and lifetime 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 the pH evolution of endosomes in real time.[12d] In this report, we investigated whether the pH response of the 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 ChemBioChem 2015, 16, 1647 – 1656

approach is motivated by the different pKa(N3) values of free 5-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] Kulikowski et 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 modifications 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 +0.14 and ¢0.22 pH units and the folding kinetics by a factor of 10, whereas the previously observed partitioning of the folding pathways remained unaltered.

Name

Sequence 5’!3’

Name

Sequence 5’!3’

I3 I3Me4

(CCCTAA)3CCC (C5mCCTAA)3C5mCC

I4 I3Br2

(CCCCTAA)3CCCC (C3TA2C5BrCCTA2C3TA2C5BrCC

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 in an identical manner compared to a polydT sequence of identical number of nucleotides (polydT T21) (Figure 1 A). Thus, introduction of bromo- or methyl-substituted cytidines into the oligonucleotides did not lead to any significant migration difference when DNA molecules were fully relaxed. As a consequence, differences in migration on native PAGE can be interpreted as arising from differences in secondary structure. The polydT sequences (dT10, dT21, and dT25) were used as size markers, assuming that their migration behaviour was not affected by differences in pH. At pH 5.0 (Figure 1 B), the sequences of interest formed species that migrated roughly together with T10, appearing twice as small as predicted from their actual

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

size. The intramolecular i-motif structure is more compact than an ssDNA coil and is expected to migrate faster than dT21 sequences without structure. All sequences showed one strong band, indicating that they formed a monomeric structure at acidic pH. Interestingly, I3Br2 migrated slightly slower than I3 and I3Me4, possibly due to formation of a less compact structure. In both gels, the I3Br2 lane showed a higher light band that might correspond to a stable dimer. Certain nucleic acid secondary structures are not fully disrupted in regular denaturing gel.[27] We further tested i-motif formation by NMR spectroscopy. The imino proton engaged in the C·C + base pair has a characteristic chemical shift around 15.5 ppm. Figure 2 shows the 1H 1D spectra of each DNA sequence of interest at acidic pH, focusing on the 16–15 ppm region. In the corresponding NOESY spectra, cross peaks could be observed between intercalated C·C + base pairs protons, caused by their close proximity (3.3 æ average distance, as determined by NMR structure 1EL2;[24] Figure S1 in the Supporting Information). This cross-peak pattern provided 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 NMR concentration. This marginal population was considered not important in following experiments. pH and thermal stability We monitored pH-dependent i-motif formation by circular dichroism (CD) spectroscopy. The resulting CD spectra acquired over the pH range 7.2 to 4.8 are presented in Figure 3. The I3Me4 and I3Br2 sequences revealed similar spectral characteristics to I3 and I4. At pH 5.0, the oligonucleotides (ODNs) displayed a maximum band around 288 nm and a minimum band between 255 and 260 nm (individual values are given in ChemBioChem 2015, 16, 1647 – 1656

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Figure 2. A) Hemiprotonated cytidine·cytidine + (C·C + ) base pairs with a 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 DNA sequences I3, I4, I3Me4, and I3Br2 at slightly acidic pH.

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

[a]

max. band [nm]

min. band[b] [nm] isoelliptic points [œ 0.2 nm]

I3

I4

I3Me4

I3Br2

288.3 (275.4) 257.3 (248.3) 277.0 246.0

287.7 (275.7) 260.3 (247.1) 278.0 243.2

288.3 (274.5) 254.7 (249.8) 276.6 n.o.

288.5 (275.2) 254.5 (248.3) 278.0 244.2

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

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, characteristic of a single-stranded DNA random coil conformation.[30] In addition, pH titration of the CD spectra of I3, I4, and I3Br2 revealed 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 nonnatural nucleotides did not impair the formation of i-motif structure. The introduction of 5-MeCs into i-motif sequences has already been studied, and similar results as reported herein have been observed.[24, 32] On the other hand, the introduction of 5-BrCs was never reported.

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

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 formation to follow the state of folding for each of the sequences over a pH range between 4.8 and 7.2. The pH-dependent folding was fully cooperative for all systems investigated. At the lowest and highest pH values, we observed maximal and minimal ME288/289, respectively. We conclude that the pH-induced transition can be well titrated over the chosen pH range. Consequently, we converted the ME288/289 into the DNA fraction folded (FF). Plots of the FF against pH values are presented in Figure 4. In order to use an i-motif as a pH sensor, the midpoint of the pH-dependent cooperative folding/unfolding transition must coincide with the (cellular) pH of interest. We defined the width of the pH transition as the pH response range of the Crich sequence. In order to compare the pH response ranges of the various i-motifs studied, we defined this within an upper and 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-

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

I3 I3Me4 I3Br2 I4

95 % folded

50 % folded

5 % folded

5.90 5.88 5.68 6.32

6.26 œ 0.01 6.40 œ 0.02 5.93 œ 0.01 6.64 œ 0.01

6.59 6.73 6.22 6.91

œ standard error from fitting in Figure 4.

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operativity of folding were apparent. The transition range (pHFF95–pHFF5) of I3 spanned 0.69 units. The pH response range of I3Br2 was narrower, with a transition range of 0.54, contrary to I3Me4, which showed a broader transition of 0.85. As a result, the introduction of 5-MeCs led to a decrease in cooperativity of the pH-induced folding transitions, contrary to what was observed upon introduction of 5-BrCs. From PAGE analysis, we demonstrated that the investigated sequences adopted an intramolecular, monomeric i-motif. On this basis, we calculated the

Figure 4. pH melting curves of i-motif sequences I3, I3Me4, I3Br2, and I4 over the pH range 4.8–7.2 at 298 K. The plots are derived from molar ellipticity at 288 nm (I3, I4, and I3Me4) or 289 nm (I3Br2), monitored during the pH titration presented in Figure 3. The CD data were transformed into folded fraction and plotted against pH values. Fitting was performed by using five points measured in triplicate. The error bar dots were obtained by averaging the values of the triplicate measures, the limits of the error bars correspond to the highest and the lowest values.

thermodynamic parameters from CD temperature denaturation curves. We measured melting curves at two different pH values. Here, it is relevant to compare the thermodynamic parameters of the sequences at a pH value within their pH response 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 resulting melting curves are presented in Figure 5. As expected, the melting temperature (Tm) of each sequence decreased as the pH increased (see Table 4). Based on the Tm values at pH 5.0, I3Me4 was the most stable i-motif compared to I3 and I3Br2, with the latter being the least stable. Interestingly, at their respective pHFF50 values, these sequences had similar Tm values.

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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 to be expressed as fraction folded.

Kinetic investigations

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

I3 I3Me4 I3Br2 I4

pH

[a]

[b]

Tm [8C]

Tm

5.0 5.0 5.0 5.0

55.7 59.1 45.5 68.4

54.8 œ 0.1 57.0 œ 0.2 44.6 œ 0.1 67.9 œ 0.2

[C]

pH 6.3 6.4 5.9 6.6

T

[a] m

[8C]

26.4 27.5 26.4 35.1

Tm

[b]

[C]

25.8 œ 0.1 27.6 œ 0.1 26.6 œ 0.1 34.1 œ 0.1

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

At pH 5.0, all sequences were folded both at 25 and 37 8C except I3Br2, of which 20 % were in random coil DNA structures at 37 8C. At pHFF50, we observed reduced stability. Indeed, at 25 8C, the i-motif populations of I3, I3Me4, and I3Br2 decreased by a third and almost completely disappeared at 37 8C. I4 presented a higher stability, due to its extra two C·C + base pairs. Consequently, considering its pHFF50, the I4 i-motif at 25 8C was largely formed, but its folded conformation decreased by two-thirds at 37 8C. Nevertheless, for I4, the quadruplex form was still populated at its pHFF50, contrary to the other sequences studied. Recently, Xu et al. investigated the introduction of one or two 5-MeCs at different positions into the I3 sequence. They reported that the position of one modified cytosine showed a limited influence on pHFF50. However, they observed a greater effect on the pH response range when both cytosines of a C·C + base pair were substituted instead of one. This observation 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 cooperativity.[33] The introduction of only one 5-MeC into I3 leads to a more modest DpHFF50,[32c] and it is possible that different parental sequences then undergo different effects due to the modified cytidine introduction. In the case of I3Br2, it was strikChemBioChem 2015, 16, 1647 – 1656

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ing that the exchange of only one C·C + base pair by a homo 5-BrC·5-BrC + base pair led to such tremendous effect on the I3 pH response range. The introduction of one 5-hydroxymethylcytosine (5-hmC) in i-motif sequences also led to an acidic tuning.[32c, 33] Yang et al. measured the base-pairing energies (BPEs) of 5xC·5-xC + homodimers by guided ion beam tandem mass spectroscopy. They revealed that 5-MeC·5-MeC + dimers comprised a higher BPE than C·C + dimers (177.4 and 169.9 kJ mol¢1, respectively).[34] In line with these findings, we propose that 5MeC·5-MeC + base pairs in i-motifs are more stable than C·C + base pairs. As a result, the pH response range might be broadened. Due to its lower pKa(N3), 5-BrC has a weaker proton affinity than C. As a consequence, the 5-BrC·5-BrC + base pair might disrupt at more acidic pH than do C·C + base pairs. However, 5-BrC·5-BrC + dimers show only a slightly lower BPE (168.5 kJ mol¢1) than the unmodified parent, which implies that both base pairs have similar stability.[34]

In previous work, we elucidated the folding kinetics pathway of the human telomeric sequence I3. We established that 1) the folding proceeds in two steps, and 2) two different folded i-motif conformations are present at equilibrium. Initially, 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 characteristic chemical 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 to 25 8C, we followed the evolution of successive 1D 1H NMR spectra over a period of 24 h. Figure 6 A shows a sample of spectra, focusing on the imino proton region, at different time points. 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. These data were then plotted against time to obtain the kinetic traces presented in Figure 6 B. Given the large effort required to assign individual resonances for the two-state population of i-motifs,[19–20, 25, 35] we decided to focus here on analysis of the kinetics without individual assignment, because the kinetic process did not reveal any differences between different nucleotides in the sequence; in other words, there were no single nucleotide-specific variations. The four strongest peaks (at 15.77, 15.56, 15.48, and 15.41 ppm) showed a constant increase before reaching a plateau. The peaks at 15.34 and 15.53 ppm showed an increase during the first 30 min before decrease and finally reached a plateau. The number of states involved in the kinetics of folding therefore remained unchanged compared to our previous studies of the I3 sequence, allowing us to conclude that the

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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 proton peaks intensities are plotted as a function of time to give kinetic traces. The grey dots correspond to the experimental data, and the orange dots correspond to 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 I4 folding pathway from the unfolded state (U) towards i-motif structures partitioned between two conformers, according to the model published by Lieblein et al.[19]

kinetics of I4 folding followed the same model as I3 (see Figure 6 C). We then studied folding of all sequences by CD spectroscopy to investigate folding kinetics in more detail. Depending on the kinetics, i-motif folding was initiated by a pH jump using an automatic mixing device (stopped-flow system) or by manual mixing. We titrated folding kinetics over the pH ramp: pHFF100, pHFF75, pHFF50, and pHFF25. Similar to the melting curves described above, the characteristic i-motif CD signature at 288 nm was monitored over time after the pH jump. After baseline and zero corrections, the molar ellipticity at 288 nm was plotted against time to obtain the kinetic traces presented in Figure S3. Most of the collected data could be fitted by single or double exponential functions. Therefore, F-tests were systematically run to compare the two possible fits for model selection. According to these statistical tests, the best fitting was always obtained by using a double exponential function. The two rate constants describing the complex folding pathway 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 decelerated when we compared a protonation change at saturating pH (~ pH 8 to 5) to a change at non-saturating pH (~ pH 8 to pHFF75, for example), as previously observed for I3.[36] In fact, it ChemBioChem 2015, 16, 1647 – 1656

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took 0.5, 10, and 4 s for I3, I3Me4, and I4, respectively, to reach the equilibrium plateau after a pH jump from 8 to 5, whereas it took more than 1000 s after a smaller amplitude pH jump. Interestingly, only I3Br2 did not show such strong effects. For I3Br2 folding, equilibrium was reached in 100 s for a pH jump towards 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 obtained for the saturating pH jump and the non-saturating pH jumps, we observed differences of a factor of 1000, 100, and 10 000 for I3, I3Me4, and I4, respectively. It is noteworthy to point out that both rate constants were affected in a similar manner for the same pH jump. In contrast, rate constants obtained 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 single exponential function was the best to describe their data.[36] According to our observations, CD spectroscopy is a reliable method to monitor the complexity of the i-motif folding mechanism, and all CD kinetic traces had to be fitted to a double exponential function. Although CD spectroscopy was unable to detect the different conformers 3’E and 5’E determined by

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Full Papers According to our data for I3, I3Me4, and I4, both folding steps were influenced by the proton concentration or, more precisely, by a pH range, described as protonation-saturating and -non-saturating. Only the I3Br2 case was different because only the first rate constant (k1) showed this behaviour. It appeared clear that the first conversion from a random coil DNA conformation to the i-motif, with preferred formation of the less 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 this second step, which involves structural rearrangement between two folded conformations, suggests that C·C + base pairs need to be deprotonated to be disrupted, and reprotonation is required for the formation of the compact i-motif. This deprotonation/reprotonation step, in turn, leads us to propose that unfolded or partially unfolded intermediates need to be involved in structural conversion. Interestingly, I3 and I4 showed similar k1 rate constants, but the k2 constant rate for I4 was tenfold smaller than for I3. This finding suggested that only the second step, but not the first folding step, was affected by the number of C·C + base pairs to be formed. This finding suggests that formation of the base pairs in the first step is simultaneous, and that conversion of the two conformers is influenced by the extra two C·C + base pairs.

Conclusion

Figure 7. Folding rate constants k1 and k2 of I3, I3Me4, I3Br2, and I4, corresponding to different pH jumps. The folding of the DNA sequences was triggered by a pH jump from pH 8 to acidic pH values by using stopped-flow mixing or manual mixing. The folding was then monitored by circular dichroism at 288 nm. The kinetics were titrated over a pH ramp composed of one protonation-saturating pH jump (pH 8 to 4.89 or 4.96) and three protonation-non-saturating pH jumps. The rate constants k1 and k2 were obtained by 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, and I3Me4 and the average of all k2 values of I3Br2 were plotted; error bars correspond to the maximum and the minimum values. *Standard error from fitting. **Maximum and minimum k2 values.

NMR spectroscopy, including single-labelled nucleotides,[19] because their optical signatures are equivalent, the partitioning of the folding pathway between the two conformers can nevertheless be determined unambiguously and characterised by two constant rates. ChemBioChem 2015, 16, 1647 – 1656

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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 oligonucleotides. NMR and CD spectroscopy showed that the chemical modifications do not prevent the studied DNAs from forming an i-motif at slightly acidic pH values. Gel electrophoresis reported the formation of only intramolecular folding. The new sequences containing 5-MeCs and 5-BrCs displayed a cooperative pH response. This behaviour makes them suitable for implementation in nanodevices. Introduction of 5-MeCs in I3Me4 decreases 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 network pH between 6 and 6.7 (I3Me4 responds over a pH range of 5.88–6.73).[17] Elongation of the C-tracts is also an interesting strategy, which was already explored,[12a] to tune the response toward more basic values. I4 can monitor a more basic pH than I3Me4, but once the pH response range is shifted, then the acidic range detectable by I3 is lost with I4. On the contrary, the introduction of 5-BrCs in I3Br2 leads to the opposite effect: the pH response range is shifted towards more acidic values. We did not observe thermal destabilising effects, due to the introduction of modified cytosine residues. Nevertheless, we found out that I3, I3Me4, and I3Br2 i-motifs are poorly populated at 37 8C at their respective transitional pH values, which makes them suboptimal for applications at physiological temperature. Thus, I4 sequences present an advantage, because they still show a large i-motif population at 37 8C.

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Full Papers Finally, we demonstrated that the partitioned folding pathway of the i-motif is conserved for all studied C-rich sequences. The introduction of 5-MeC does not change the folding kinetic rate, compared to I3. On the contrary, 5-BrC accelerates the folding kinetics. Elongation of the C-tracts leads to a decrease in the folding rate, due to a much slower conformer conversion during the second step of the folding mechanism. As a consequence, I4 needs hours to reach equilibrium. I3 analogue I3Me4 showed that the introduction of 5-MeC slightly decreased the cooperativity of folding and broadened the pH response range toward more basic values (+ +0.19). We could imagine that the introduction of 5-MeCs in I4 would produce 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 the design of DNA sequences that could monitor neutral and slightly basic pH conditions. Similarly, we could transfer to 5BrC-I4 analogues the properties observed for I3Br2. We showed that 5-BrC introduction shifted the pH response range of the parental sequence towards more acidic values and accelerated the folding kinetics. The resulting pH response range of a 5-BrC-I4 analogue might overlay the I3-responsive pH range. 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 applied to nanodevices. The comprehensive biophysical analysis presented here shows that it is paramount to perform a complete analysis with thermal stability and kinetic investigations of i-motif sequences for optimisation in their application as cellular nanodevices. We decided to target C·C + base pairs in our tuning tactics, but the analysis of i-motif sequences found in promotor sequences, as in bcl-2 or c-myc, suggests that long loops induce a pH stabilisation effect.[29] The loops are suspected to have a capping effect. The design of the loops in order to optimise the formation of stacking of loop nucleotides with a C·C + -cytidine core, by using Watson–Crick (WC) or non-WC base pairing into the loops, represents an interesting direction to follow. The 5’- and 3’-end regions could also be exploited to introduce supplementary stabilising elements. For instance, Nesterova et al. shifted the transitional pH of the ODN d[(C5T3)3C5] from 6.9 to 7.2 by introducing a C-rich sequence in the loop of a hairpin, with the stem of the hairpin leading to the stabilisation 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 derivatives with regard to stability and folding kinetics, will provide key insights for applications in bio-nanotechnology and beyond for optimising sequence–response relationships for this exciting class of tuneable oligonucleotides.

a 3 kDa cut-off (Vivaspin 2, Sartorius). For the I4 ODN, a LiClO4/acetone precipitation was performed to replace DNA counterions from the HPLC step with lithium, and the ODN was then dissolved in water. DNA concentrations were determined by UV/Vis spectroscopy on a Cary50 UV-spectrophotometer by using extinction coefficients at 260 nm (e260), as presented in Table 5.

Experimental Section

CD spectroscopy methods: The CD spectra and temperature melting curves were recorded on a Jasco J-810/815 CD spectropolarimeter equipped with a Jasco PTC-4235L Peltier thermostated cell holder. The cell chamber was flushed with a constant nitrogen flow to avoid water condensation on the measurement cuvette.

DNA oligonucleotides: Oligonucleotides were purchased from Eurofins MWG Operon. After HPLC purification, DNA samples were freeze-dried and desalted by using microconcentrators with ChemBioChem 2015, 16, 1647 – 1656

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

Name

Sequence 5’!3’

I3 I4 I3Me4 I3Br2

(CCCTAA)3CCC (CCCCTAA)3CCCC C5mCCTA2C5mCCTA2C5mCCTA2C5mCC C3TA2C5BrCCTA2C3TA2C5BrCC

e260 [m cm¢1] 185 900[a] 214 700[a] 180 450[b] 178 830[b]

[a] Calculated by using the nearest-neighbour model. [b] Calculated by using the base composition method. For 5-MeC and 5-BrC, e260 values of 5.7 and 3.1 mL mmol¢1, respectively, were used.

Sample preparation: Samples for circular dichroism (CD) spectroscopy experiments had DNA concentrations of 19.5–20 mm for I3, I4, I3Me4, and I3Br2, and all samples were prepared from the same stock solution. The oligonucleotides were buffered by 25 mm potassium acetate buffer over the pH range 4.8–5.6, or by 25 mm potassium phosphate buffer over a pH range of 5.8–7.2. The samples were incubated at 95 8C for 10 min and left at 4 8C at least one day for equilibration before measurement. Samples for static NMR spectroscopy were systematically snapcooled before measurement. DNA concentrations were 2 mm (I3) 600 mm (I4), 150 mm (I3Br2), and 70 mm (I3Me4). The samples were buffered by 25 mm potassium phosphate buffer at pH 5.3 (I3 and I3Br2) or 5.5 (I4 and I3Me4). The samples were supplemented with 10 % of D2O, and DSS was used as an internal reference. The I4 NMR sample for kinetics measurement was composed of 300 mm I4 DNA, 25 mm potassium phosphate buffer (pH 6.4), and 10 % D2O; DSS was used as an internal reference. The sample was incubated 5 min at 95 8C immediately prior to acquisition. Denaturing PAGE: 20 % polyacrylamide gel was mixed with 1 Õ TBE buffer pH 8.3 and urea (8 m). I3, I4, I3Me4, I3Br2, T10, T21, and T25 (150 mm each) were combined with loading buffer (99 % formamide, 0.01 % bromophenol) and loaded on the gel. The gel was run at room temperature in 1 Õ TBE buffer with a current of 220 V applied. Band migration was revealed on a silica gel plate under UV light shadowing and photographed with a digital camera. Native PAGE: 20 % polyacrylamide gel was prepared by using 1 Õ TAE buffer, pH 5.0, 4 8C. DNA samples were combined with loading buffer (50 % glycerol, 5 Õ TAE buffer, pH 5.0, 4 8C). All samples were incubated at 95 8C for 10 min and stored overnight at 4 8C before loading. The gel was run at 4 8C in 1 Õ TAE buffer with a current of 60 V applied. DNA bands were visualised on a silica gel plate under UV light, and the gel was then photographed with a digital camera.

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Full Papers For all measurements, a CD quartz cuvette with a path length of 1 mm was used. The CD spectra were recorded at 25 8C over a spectral window of 220–330 nm, with data sampling of 0.2 nm at a scan speed of 50 nm min¢1. Spectra were the result of the average of three successive acquisitions. A baseline correction was applied by using an adequate buffer solution. Only the points at critical pH values (that is, plateaus, inflection points, and transitional points) were measured in triplicate on independent samples to obtain error bars. The ellipticity values were transformed into molar ellipticity, according to Equation (1): ½q¤ ¼ ðq   MÞ=ðc   l   10Þ

ð1Þ

sented 61 spectra in total. The same baselines and t0 point were recorded as described for stopped-flow CD kinetics measures. Each kinetics experiment was performed once. After baseline and zero corrections, the ellipticity was converted into ME. The ME values at 287.5, 288.0, and 288.5 nm were averaged and plotted against time. Traces presented in Figure S4 were fitted to a 4-parameter or 5parameter double exponential function respectively, in SigmaPlot 12.5: 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

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]. The molar ellipticity values at 288 nm (I3, I3Me4, and I4) or 289 nm (I3Br2) for each pH point were extracted and normalised to be expressed as the fraction folded (1 or FF). Data for the triplicate points were fitted with a three-parameter sigmoidal function: f = a/ (1 + exp(¢(x¢x0)/b)) by using SigmaPlot 12.5 software. x0 corresponds to the transitional pH. Temperature denaturing curves were obtained by monitoring the CD at 288 or 289 nm along a temperature gradient of 4–95 8C, at a rate of 0.5 8C min¢1. One data point was recorded every 0.5 8C. The measured data were normalised and expressed as fraction folded. We determined the melting temperature by the median line method[38] and by fitting. Stopped-flow CD: SFCD measurements were carried out on a Pistar-180 system (Applied Photophysics) set up for CD detection. DNA sample solutions (10 mm DNA in buffer A: 45 mm KCl, 2.5 mm K2PO4, pH 8.02) were rapidly mixed with buffer solution (buffer B: 25 mm K2HPO4 at different pH values) in a 1:1 ratio. The mixture creates a pH jump in the direct environment of DNA molecules from basic to acidic conditions. All acquisitions were performed at 25 8C through a path length set at 10 mm. The K + cation concentration was kept constant before and after mixing (50 mm). CD evolution was monitored as function of time at 288 nm with a bandwidth of 8 nm. Kinetics traces were recorded over different periods, according to the ODN and the conditions (2–1000 s). Ten thousand points were recorded for each trace, independent of the acquisition time. Each condition was recorded five times (for traces Š 200 s) or ten times (for traces < 200 s) and then averaged. A t0 point was recorded for each condition by mixing DNA solution against buffer A. Baseline corrections (buffer A against buffer A, and buffer A against buffer B) were performed for each condition and the t0 point, according to the same parameters and repetition number of the corresponding pH jump experiment. The ellipticity was baseline and zero corrected before being converted into molar ellipticity (ME), as defined previously. In order to improve the signal/noise ratio, we averaged the ME of five successive time points, (except for I3 kinetics at pHFF75 and pHFF50) and plotted the result against time. The kinetics of I4 at pHFF75, pHFF50, and pHFF25 were recorded on the same “normal” CD spectropolarimeter as describe above. The mixing was manually performed, which led to a dead time of 15 s before the beginning of the measure. The temperature was set at 25 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 repreChemBioChem 2015, 16, 1647 – 1656

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An F-test was systematically run to define the best fitting function. Static NMR spectroscopy: Spectra were recorded on a Bruker 600 MHz (I3, I3Br2, and I3Me4) or a 950 MHz (I4) spectrometer equipped with a cryogenic probe at 298 or 288 K (I3). The 1H 1D spectra used jump-and-return for water suppression,[39] with a jump-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 monitored by real-time NMR spectroscopy on a Bruker 600 MHz spectrometer equipped with a cryogenic probe at 298 K. The sample was incubated for 5 min at 95 8C, just before acquisition of a pseudo-2D experiment with a jump-and-return sequence for water suppression.[39] This pulse sequence recorded successive 1D 1H spectra at several time points. The jump-and-return delay time was set to 25 ms, the carrier frequency in the proton dimension was set to the water frequency, and the repetition delay was 1 s. A total of 16 384 1D spectra were recorded with 5.3 s per spectrum, corresponding to an accumulation of four scans. After the five denaturing 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 with SigmaPlot 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 imino peaks, and the coefficients b and d corresponded to two rate constants (k1 and k2) describing kinetic partitioning.[19]

Acknowledgements The authors thank Dr. Boris Fìrtig and Irene Bessi for insightful discussion and Dr. Alexey Cherepanov for stopped-flow CD support. H.S. is member of the DFG-funded cluster of excellence: macromolecular complexes. BMRZ is supported by the state of Hessen. 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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Tuning the pH Response of i-Motif DNA Oligonucleotides.

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 p...
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