Analysis of the i motif structures during biased unfolding

Analysis of the i-motif structures during biased unfolding of G-quadruplexes leads to the conclusion that at acidic pH the i-motif is absolutely unaffected by that process. The hydrogen bonds within the i-motif are intact (Fig. 3D) and they only fluctuate around mean values. On the other hand, the i-motif at the neutral pH quickly lost its structure and most of hydrogen bonds become cleaved (particularly the outermost ones) as a result of the G-quadruplex unfolding. However, it seems that the obtained weakened i-motif structure is still stable as the distances only fluctuate around their mean values. On the other hand, the deterioration of the i-motif at the neutral pH was observed in the unbiased calculations but in the case of i-motif formed within the single stranded C-rich chain. [31] The presence of the complementary G-rich chain with the G-quadruplex formed needs crossing of ca. 70 kJ mol−1 of free Compstatin barrier in order to unfold the i-motif to hairpin. The enforced unfolding of G-quadruplex does not seem to significantly destabilize the complementary i-motif. Fig. 4 shows how the distances between atoms forming hydrogen bonds change during enforced unfolding of i-motif parts of the iG structures. The organization of Fig. 4 is the same as Fig. 3 so we can directly observe the differences in the unfolding processes when the bias is added to the i-motif or to G-quadruplex parts of the iG systems. Thus, we can see that during enforced unfolding of the i-motifs the associated G-quadruplexes remain almost intact (note that the bias acts up to 20 ns). The i-motifs obviously undergo unfolding but it is clearly seen that pH affects the unfolding processes significantly. At the neutral pH all hydrogen bonds break at the very beginning (though some of them are already broken after the equilibration stage) and this means that the i-motif deteriorates as a whole structure. At the acidic pH the process proceeds in a different way. The deterioration of the i-motif begins from the middle pair of cytosines and next the outermost pairs undergo cleavage. The innermost pairs are the most resistant and they partially recover after removing of the biasing forces. In this case the i-motif seems to tend to hairpin form (see Fig. 2) contrary to the neutral pH case when the i-motif is spatially destroyed and all the distances from Fig. 4 are above 10 Å. The enforced unfolding of the i-motifs do not affect the complementary G-quadruplexes, as seen in Fig. 4A and C. However, after removing the biasing force something unexpected happens to G-quadruplex at the neutral pH. Namely, the relaxation of the i-motif part seems to induce some deterioration of the G-quadruplex. Precisely, the hydrogen bonds between the innermost quartet of guanines break though the distances do not grow strongly. Moreover, visual inspection of the spatial structure of the G-quadruplex (Fig. 2, part In) does not show a significant deformation or deterioration of the G-quadruplex in this case. Thus, the observed increase of the distances in Fig. 4A occurred probably only incidentally and this is simply a transient state from which the hydrogen bonds spontaneously recover after some time. Analysis of the work done during the enforced unfolding of both the i-motif and the G-quadruplex allows us to draw quantitative conclusions concerning the stabilities of these structures. Fig. 5 shows the work associated with the forces generated by the linearly increasing (with the constant velocity 1.25 Å ns−1) rmsd in the case of the G-quadruplex unfolding. As seen in Fig. 5 in both cases of pH the work necessary to destroy the G-quadruplexes are very similar. The curves reveal stepwise shape with the most visible platteau within 3–5 Å of the rmsd (or 2.6–4 ns of the simulation time). The height of that first step is ca. 250 kJ mol−1 and this is associated with the cleavage of 3 hydrogen bonds, as seen in Fig. 3. However, in spite of the similarity between the plots in Fig. 5 the mechanism of the unfolding is different in these two cases. According to Fig. 3 the unfolding starts from the innermost guanine ring at the neutral pH and from the outermost in the case of the acidic pH. However, the energetic effect is very similar in both cases though it cannot be associated only with the hydrogen bonds cleavage. This is because the energies are much bigger than 3 times the energy of a single hydrogen bond, which is ca. 20 kJ mol−1. [19]