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![Denaturation profiles obtained in a 10 mM sodium cacodylate pH 6.4 buffer for the 18(O) (open circles) and 18(S) (black triangles) d(CCTTTCCTT- TACCTTTCC) oligodeoxynucleotides (6 µM strand concentration). The cooling and heating profiles were superimposable at this pH. (A) 265 nm. (B) 295 nm. (C) Thermodynamic analysis of the denaturation profile: Van't Hoff plot of ln(K) versus 1/T [18(O): open circles; 18(S): black triangles]. K is the equilibrium association constant. The fraction of oligodeoxynucleotide engaged in i-motif formation was determined from the profiles at 295 nm.](profile/Jean-Louis-Mergny/publication/13508950/figure/fig1/AS:349532085866520@1460346351974/Denaturation-profiles-obtained-in-a-10-mM-sodium-cacodylate-pH-64-buffer-for-the-18O.png)
Denaturation profiles obtained in a 10 mM sodium cacodylate pH 6.4 buffer for the 18(O) (open circles) and 18(S) (black triangles) d(CCTTTCCTT- TACCTTTCC) oligodeoxynucleotides (6 µM strand concentration). The cooling and heating profiles were superimposable at this pH. (A) 265 nm. (B) 295 nm. (C) Thermodynamic analysis of the denaturation profile: Van't Hoff plot of ln(K) versus 1/T [18(O): open circles; 18(S): black triangles]. K is the equilibrium association constant. The fraction of oligodeoxynucleotide engaged in i-motif formation was determined from the profiles at 295 nm.
Source publication
At slightly acidic or even neutral pH, oligodeoxynucleotides that include a stretch of cytidines have been shown to form a
tetrameric structure in which two parallel-stranded duplexes have their hemiprotonated C.C+ base pairs face to face and fully intercalated, in a so-called i-motif. Cytosine-rich pyrimidine oligodeoxynucleotides can
form an intr...
Contexts in source publication
Context 1
... of the i-motif leads to an hyperchromism at 265 nm (3,10,19). An example of a denaturation/renaturation profile at pH 6.4, obtained for the 18(O) and 18(S) pyrimidine oligo- deoxynucleotides d-CCTTTCCTTTACCTTTCC is presented in Figure 1. A denaturation profile is characterized by a sharp increase of absorbance at 265 nm (Fig. 1A), whereas a hypochromic transition is observed at 295 (Fig. 1B) (19) or 305 nm (18). ...
Context 2
... of the i-motif leads to an hyperchromism at 265 nm (3,10,19). An example of a denaturation/renaturation profile at pH 6.4, obtained for the 18(O) and 18(S) pyrimidine oligo- deoxynucleotides d-CCTTTCCTTTACCTTTCC is presented in Figure 1. A denaturation profile is characterized by a sharp increase of absorbance at 265 nm (Fig. 1A), whereas a hypochromic transition is observed at 295 (Fig. 1B) (19) or 305 nm (18). Both curves could well be analysed as an all-or-none intramolecular phenomenon. Thermodynamic parameters determined from the profiles at 265 and 295 nm were identical within experimental error. The difference between the absorbance spectra of cytosine ...
Context 3
... at 265 nm (3,10,19). An example of a denaturation/renaturation profile at pH 6.4, obtained for the 18(O) and 18(S) pyrimidine oligo- deoxynucleotides d-CCTTTCCTTTACCTTTCC is presented in Figure 1. A denaturation profile is characterized by a sharp increase of absorbance at 265 nm (Fig. 1A), whereas a hypochromic transition is observed at 295 (Fig. 1B) (19) or 305 nm (18). Both curves could well be analysed as an all-or-none intramolecular phenomenon. Thermodynamic parameters determined from the profiles at 265 and 295 nm were identical within experimental error. The difference between the absorbance spectra of cytosine and protonated cytosine explains the inverted denaturation ...
Context 4
... nm (18). Both curves could well be analysed as an all-or-none intramolecular phenomenon. Thermodynamic parameters determined from the profiles at 265 and 295 nm were identical within experimental error. The difference between the absorbance spectra of cytosine and protonated cytosine explains the inverted denaturation curves obtained at 295 nm (Fig. 1B). The shorter wavelength (265 nm, close to the isosbestic point of the pH titration) gives information which is independent of the protonation state of the cytosines. The longer wavelength (295 nm) should tell us whether the formation of a structure requires protonation/ deprotonation of some cytosines: at this wavelength, the ...
Context 5
... the equilibrium association constant K of the i-motif, can be plotted against 1/T. As shown in Figure 1C, an excellent linear fit can be derived from the experimental points (r > 0.997), showing that the data is in excellent agreement with an all-or-none simple intramolecular equilibrium. However, one cannot fail to notice that the slopes of the curves are different, thus leading to different ∆H_ values [∆H_ = -60 kcal/mol for the 18(O) oligodeoxynucleotide and ∆H_ = -48 kcal/mol for the 18(S) oligodeoxynucleotide]. ...
Context 6
... with PO homologs, as shown by a nearly identical melting temperature at various pHs. Intramolecular folding was demonstrated by the concentration independence of their melting profiles in the micromolar concentration range. Nevertheless, i-motif formation for these analogs was significantly less enthalpy driven [as shown for the 18(S) oligo in Fig. 1C]. Depending on the orientation of critical phosphorothioate groups, a mixture of different i-motif isomers with slightly different stabilities might be obtained, thus leading to a global melting curve reflecting the algebraic sum of a number of different equilibria. This phenomenon, which was described previously for PS duplexes, may ...
Context 7
... on the orientation of critical phosphorothioate groups, a mixture of different i-motif isomers with slightly different stabilities might be obtained, thus leading to a global melting curve reflecting the algebraic sum of a number of different equilibria. This phenomenon, which was described previously for PS duplexes, may explain the lower absolute value of the enthalpy of formation, resulting in apparently 'less cooperative' melting processes, such as the one presented in Figure 1. Despite this slightly lower ∆H_, PS and PO oligodeoxynucleotides showed similar T m s, showing that the oxygen to sulfur substitution had little effect on the thermodynamic stability of the i-motif. ...
Citations
... Both these fields clearly require a better understanding of the properties of these non-canonical arrangements and several studies have been devoted at identifying nucleoside modifications that might enhance their stability (in particular under neutral conditions) to be used as probes in cells or as biosensors [20]. The substitution of the phosphodiester backbone with peptide nucleic acid (PNA) or phosphorothioate (PS) linkages did not provide relevant improvements [21,22]. More promising results were obtained with nucleobase variations. ...
I-motifs are non-canonical DNA structures consisting of two parallel strands held together by hemiprotonated cytosine-cytosine+ base pairs, which intercalate to form a ordered column of stacked base pairs. This unique structure covers potential relevance in various fields, including gene regulation and biotechnological applications. A unique structural feature of I-motifs (iM), is the presence of sugar-sugar interactions through their extremely narrow minor grooves. Consistently, oligonucleotides containing pentose derivatives such as ribose, 2'-deoxyribose, arabinose, and 2'-deoxy-2'-fluoroarabinose highlighted a very different attitude to fold into iM. On the other hand, there is significant attention focused on exploring sugar-modifications that can increase nucleic acids resistance to nuclease degradation, a crucial requirement for therapeutic applications. An interesting example, not addressed in the iM field yet, is represented by hexitol nucleic acid (HNA), a metabolically stable six-membered ring analogue compatible with A-like double helix formation. Herein, we selected two DNA C-rich Tetrahymena telomeric sequences whose tetrameric iMs were already resolved by NMR and we investigated the iM folding of related HNA and RNA oligonucleotides by circular dichroism, differential scanning calorimetry and NMR. The comparison of their behaviours vs the DNA counterparts provided interesting insights into the influence of the sugar on iM folding. In particular, ribose and hexitol prevented iM formation. However, by clustering the hexitol-containing residues at the 3'-end, it was possible to modulate the distribution of the different topological species described for the DNA iMs. These data open new avenues for the exploitation of sugar modifications for I-motif characterization and applications.
... Kinetics of iM formation has been extensively studied in a number of papers, e.g. (6,7,40,46). For the telomeric DNA sequence (6,7) 3´E and 5´E iM topologies are formed (upon transfer to acidic environments) with fast and slow kinetics, respectively, while the 5´E gradually replaces the kinetically driven 3´E structure. ...
Cytosine-rich DNA regions can form four-stranded structures based on hemi-protonated C.C+ pairs, called i-motifs (iMs). Using CD, UV absorption, NMR spectroscopy, and DSC calorimetry, we show that model (CnT3)3Cn (Cn) sequences adopt iM under neutral or slightly alkaline conditions for n > 3. However, the iMs are formed with long-lasting kinetics under these conditions and melt with significant hysteresis. Sequences with n > 6 melt in two or more separate steps, indicating the presence of different iM species, the proportion of which is dependent on temperature and incubation time. At ambient temperature, kinetically favored iMs of low stability are formed, most likely consisting of short C.C+ blocks. These species act as kinetic traps and prevent the assembly of thermodynamically favored, fully C.C+ paired iMs. A higher temperature is necessary to unfold the kinetic forms and enable their substitution by a slowly developing thermodynamic structure. This complicated kinetic partitioning process considerably slows down iM folding, making it much slower than the timeframes of biological reactions and, therefore, unlikely to have any biological relevance. Our data suggest kinetically driven iM species as more likely to be biologically relevant than thermodynamically most stable iM forms.
... T m in this work is defined as the average of T 1/2 obtained from heating and cooling processes. 10,54 An excellent negative linear correlation (Pearson's r > 0.99) between T m and pH was found for all sequences in this pH range ( Figure 5B). Linear fitting results of T m as a function of pH are given in Table S2. ...
... In addition, past a given length of helix, certain approximations such as two-state melting are no longer usable. 59,60 Second, hysteresis between the thermal denaturation and renaturation processes has been observed, 5,10,11,13,20,61 and hysteresis between pH denaturation and renaturation processes has also been found. 12,61 This is the first report in which hysteresis for both thermal and pH transitions is analyzed simultaneously. ...
... When a hysteresis between heating and cooling profiles is present (T 1/2,heating ≠ T 1/2,cooling ), both folding and unfolding process are relatively slow, and we used the average value between T 1/2 from heating and cooling processes as an approximation for the equilibrium melting temperature, T m . 53,54,85 This averaging method actually gives an excellent estimate of the real T m , as illustrated in ref 10. ...
... Then thermal denaturation of i-DNAs at pH 5.0 and pH 7.0 was tracked by UV-absorbance at 295 nm (Figures S16-S18). [23] At neutral pH, only sequences with longer Ctracts such as C 5 and C 6 were considered, as sequences with shorter C-tracts do not fold or exhibit low stabilities (T m < 12 8 8C) preventing accurate determinations.F olding and unfolding processes follow relatively fast kinetics under [a] Counts based on results presented in Tables S1,S2, Figures S9 and S14. The thermal stability of sequences with C 3 and C 4 -tracts at pH 7.0 was not evaluated. ...
... [5b] Hysteresis is determined for each melting/annealing experiment described in this paper,and T m average between cooling and heating was taken as ap roxy for thermodynamic stability,a s previously found for other i-DNAs tructures. [23] Forp H T determination, each sample was allowed to anneal at agiven pH for al ong period (> 12 hours), allowing thermodynamic equilibrium. ...
Recent studies indicate that i‐DNA, a four‐stranded cytosine‐rich DNA also known as the i‐motif, is actually formed in vivo; however, a systematic study on sequence effects on stability has been missing. Herein, an unprecedented number of different sequences (271) bearing four runs of 3–6 cytosines with different spacer lengths has been tested. While i‐DNA stability is nearly independent on total spacer length, the central spacer plays a special role on stability. Stability also depends on the length of the C‐tracts at both acidic and neutral pHs. This study provides a global picture on i‐DNA stability thanks to the large size of the introduced data set; it reveals unexpected features and allows to conclude that determinants of i‐DNA stability do not mirror those of G‐quadruplexes. Our results illustrate the structural roles of loops and C‐tracts on i‐DNA stability, confirm its formation in cells, and allow establishing rules to predict its stability.
... Hypochromicity at 295 nm is a characteristic of i-motif unfolding, 9 which has been widely adopted to monitor i-motif's denaturing process. 10,11,12,13,14 pH Denaturation studies by circular dichroism (CD). Prior to circular dichroism (CD) measurement, a solution of the C-rich DNA (end concentration of the two-C-tracts fragment was 5.0 µM, 0.4 mL) was prepared in citric acid-Na 2 HPO 4 buffer (20 mM, pH 5.3-7.9) with 100 mM NaCl, and was heated to 85 °C, then cooled to room temperature with a gradient of 1.0 °C/min. ...
The response sensitivity of a molecular sensor is determined by the folding cooperativity of its responsive module. Using H+-responsive dimeric DNA i-motif as a model, we demonstrate the enhancement of...
... Then thermal denaturation of i-DNAs at pH 5.0 and pH 7.0 was tracked by UV-absorbance at 295 nm (Figures S16-S18). [23] At neutral pH, only sequences with longer C-tracts such as C5 and C6 were considered, as sequences with shorter C-tracts do not fold or exhibit low stabilities (Tm < 12 °C) preventing accurate determinations. Folding and unfolding processes follow relatively fast kinetics under mildly acidic conditions, as expected for intramolecular folding. ...
... [5b]5b] Hysteresis is determined for each melting/annealing experiment described in this paper, and Tm average between cooling and heating was taken as a proxy for thermodynamic stability, as previously found for other i-DNA structures. [23] For pHT determination, each sample was allowed to anneal at a given pH for a long period (> 12 hours), allowing thermodynamic equilibrium. ...
Recent studies indicate that i‐DNA, a four‐stranded cytosine‐rich DNA also known as the i‐motif, is actually formed in vivo ; however, a systematic study on sequence effects on stability has been missing. Herein, an unprecedented number of different sequences (271) bearing four runs of 3‐6 cytosines with different spacer lengths has been tested. While i‐DNA stability is nearly independent on total spacer length, the central spacer plays a special role on stability. Stability also depends on the length of the C‐tracts at both acidic and neutral pHs. This study provides a global picture on i‐DNA stability thanks to the large size of the introduced data set; it reveals unexpected features and allows to conclude that determinants of i‐DNA stability do not mirror those of G‐quadruplexes. Our results illustrate the structural roles of loops and C‐tracts on i‐DNA stability, confirm its formation in cells, and allow establishing rules to predict its stability.
... Importantly, we analysed kinetics of pH-driven iM folding/unfolding in connection with thermodynamics. While the link between kinetics and thermodynamics of temperature-driven iM-ssDNA transitions is well-known (Mergny and Lacroix, 1998), the interdependence of iM responses to temperature and pH stimuli has not been discussed explicitly so far. We revealed a correlation between the rates of these responses. ...
We report the design of robust sensors for measuring intracellular pH, based on the native DNA i-motifs (iMs) found in neurodegeneration-or carcinogenesis-related genes. Those iMs appear to be genomic regulatory elements and might modulate transcription in response to pH stimuli. Given their intrinsic sensitivity to minor pH changes within the physiological range, such noncanonical DNA structures can be used as sensor core elements without additional modules other than fluorescent labels or quenchers. We focused on several iMs that exhibited fast folding/unfolding kinetics. Using stopped-flow techniques and FRET-melting/annealing assays, we confirmed that the rates of temperature-driven iM-ssDNA transitions correlate with the rates of the pH-driven transitions. Thus, we propose FRET-based hysteresis analysis as an express method for selecting sensors with desired kinetic characteristics. For the leading fast-response sensor, we optimized the labelling scheme and performed intracellular calibration. Unlike the commonly used small-molecule pH indicators, that sensor was transferred efficiently to cell nuclei. Considering its favourable kinetic characteristics, the sensor can be used for monitoring proton dynamics in the nucleus. These results argue that the 'genome-inspired' design is a productive approach to the development of biocompatible molecular tools.
... A few studies on iM folds have identified and begun to understand folding hysteresis for specific sequences. [12,26] Studies to compare different analytical methods to yield iM-specific parameters and find experimental dependency have not been reported; inspection of iM hysteresis is, therefore, ripe for further study. ...
... Consistent with this finding is prior work from our laboratory and others that found other iMs produced isothermal hysteresis in the pH T values. [12,19,26] The more interesting finding in the present studies is that the method (1 vs 2) for titrating iM-forming sequences to measure pH T values can yield different results. The difference was maximally displayed in the dC 15 and dC 19 sequences (Figure 2A,B). ...
In DNA, i‐motif (iM) folds occur under slightly acidic conditions when sequences rich in 2′‐deoxycytidine (dC) nucleotides adopt consecutive dC self base pairs. The pH stability of an iM is defined by the midpoint in the pH transition (pHT) between the folded and unfolded states. Two different experiments to determine pHT values via circular dichroism (CD) spectroscopy were performed on poly‐dC iMs of length 15, 19, or 23 nucleotides. These experiments demonstrate two points: (1) pHT values were dependent on the titration experiment performed, and (2) pH‐induced denaturing or annealing processes produced isothermal hysteresis in the pHT values. These results in tandem with model iMs with judicious mutations of dC to thymidine to favor particular folds found the hysteresis was maximal for the shorter poly‐dC iMs and those with an even number of base pairs, while the hysteresis was minimal for longer poly‐dC iMs and those with an odd number of base pairs. Experiments to follow the iM folding via thermal changes identified thermal hysteresis between the denaturing and annealing cycles. Similar trends were found to those observed in the CD experiments. The results demonstrate that the method of iM analysis can impact the pHT parameter measured, and hysteresis was observed in the pHT and Tm values.
Abstract
... This structure is formed with two parallel duplexes linked via hemi-protonated cytosine-cytosine base pairs that are intercalated in an antiparallel orientation [37,38] (Figure 9). cytosine [39] , loop length [40] , or environmental condition. Indeed, it has been shown that imotif can even be formed at neutral pH conditions [41] and supports the idea that i-motif are dynamic structure over a wide range of pH from a folded structure to a more disorganized conformation especially at higher pH values. ...
KRAS gene codes for a highly mutated GTPase protein acting as a « switch » between an active and an inactive state, a mechanism found to be important in biological processes such as cell replication and proliferation. When misregulated, these processes are found to be at the origin many types of cancer. KRAS mutations are particularly implicated in lung (30%), colorectal (44%) and pancreatic (97%) cancers. Despite the fact that those mutations are well known, KRAS is still an undruggable target because all the actual strategies (RAS activator inhibitors, membrane association inhibitors, and so on) are not efficient enough as cancer therapies. That is why new strategies have emerged recently, such as directly targeting the KRAS promoter region and especially some specific structures called G-quadruplexes (G4). Although we do not understand well the phenomena, there are plenty of evidence in the literature that these structures can assemble both in vitro and in cellular conditions. It was shown that G4 within KRAS promoter region can bind transcription related proteins and disturb transcription process acting as a block mechanism when transcription machinery is reading the genetic sequence. Stabilization of these structures, using small chemical ligands for example, could become a new area of therapy. In my thesis work, I am focused on a 32 nucleotide sequence (KRAS32R) which can form G4 and also corresponds to the minimal interaction domain of transcription proteins such as MAZ or hnRNP1. This last protein is capable of binding to KRAS32R G-quadruplexes and possibly unfolding it, favoring the transcription of KRAS. This project aims to understand the folding of this KRAS32R G-quadruplex at atomic level and its interaction with small organic molecules that would have some effect on transcription process.
... In an attempt to suppress the repulsion between the negatively charged phosphate backbones, several backbone modifications have been investigated. Mergny and Lacroix investigated the effect of phosphorothioate, and methylphosphonate, as opposed to the phosphodiester backbone (47). Their studies show that only backbones exhibiting phosphodiester and phosphorothioate bonds allow imotif formation. ...
... Additionally, the chirality of the methylphosphonate linkage (presence of both Rp and Sp stereochemistries) might have influenced i-motif formation and stability. The incorporation of phosphorothioates in several DNA C-rich sequences leads to the formation of stable i-motif structures at neutral pH, and they are only a few degrees less stable than the unmodified structures (47). Moreover, the chirality of the phosphorothioate group influences i-motif stability; for instance the Rp-stereochemistry leads to greater stabilization compared to the Sp-stereochemistry ( T m = 11 • C) (48). ...
The i-motif represents a paradigmatic example of the wide structural versatility of nucleic acids. In remarkable contrast to duplex DNA, i-motifs are four-stranded DNA structures held together by hemi- protonated and intercalated cytosine base pairs (C:C+). First observed 25 years ago, and considered by many as a mere structural oddity, interest in and discussion on the biological role of i-motifs have grown dramatically in recent years. In this review we focus on structural aspects of i-motif formation, the factors leading to its stabilization and recent studies describing the possible role of i-motifs in fundamental biological processes.
