The stability of the i motif at the
The stability of the i-motif at the neutral pH has been analyzed in our previous paper  by tracking the rmsd changes in unbiased calculations. Fig. 7 presents, just to confirm the conclusions drawn in , the changes in distances between atoms forming hydrogen bonds within the i-motif structures at both acidic and neutral pH. Fig. 7 shows actually analogous results like Fig. 3, Fig. 4 but it concerns the iG structures without any bias added. So, the conclusions from Fig. 7 are obvious for the acidic pH. Namely, the i-motif is kept by many hydrogen bonds and it does not deteriorate within quite long simulation time reaching 50 ns. However, a few bonds break occasionally but they recover after some time. This happens for the bonds localized at the bottom of the i-motif. At the neutral pH the i-motif is, as mentioned, weakened and this is confirmed by the distances shown in Fig. 7. Many hydrogen bonds disappeared as the distances are bigger than 3.5 Å, however there is still large number of distances around 3 Å which indicate the presence of hydrogen bonds. But the most important observation is that the distances fluctuate but do not grow above 20 Å. This means that the i-motif spatial shape is fairly well preserved at the neutral pH and it does not unfold totally. In ref.  we concluded that the i-motif at the neutral pH is stabilized by the presence of the G-quadruplex in the complementary GSK1210151A synthesis rich strand. However, the current study leads to some revision of the role of G-quadruplex in the stabilization of the i-motif. Fig. 8A shows the results concerning the enforced unfolding of i-motifs but currently the starting and reference structures are the final states obtained after unfolding of the G-quadruplexes. These reference states are the Ga and Gn structures from Fig. 2 and the biases are added to i-motif parts in order to study unfolding of i-motifs when the G-quadruplexes do not exist any more. Similarly, Fig. 8B shows work measured during enforced unfolding of G-quadruplexes from the states where i-motifs have already been unfolded, i.e. states Ia and In from Fig. 2. As seen in Fig. 8A the lack of G-quadruplex at acidic pH does not affect the stability of the i-motif. It is highly stable no matter whether there is the G-quadruplex formed in the complementary guanine rich strand or that part of the strand is in structural disorder. At the neutral pH, however, we observe quite surprising result. Namely, it seems that the structural disorder in the guanine rich strand stabilizes the i-motif when compared to the previous case of the presence of a perfect G-quadruplex structure. The work necessary to unfold the i-motif is significantly larger than that corresponding to the unfolding of i-motif from the ideal iG structure. Fig. 9 shows two simulation snapshots taken at rmsd 5 Å and 15 Å which help to understand why the presence of the unfolded G-quadruplex fragment stabilizes the i-motif stronger than the ideal G-quadruplex structure. As seen in Fig. 9 the i-motif is able to approach the spatially extended structure formed by the duplex fragment and the unfolded G-quadruplex. This enhances the pairwise interactions between the i-motif forming atoms and these belonging to the residues which formed G-qudruplex and duplex. Mutual interactions between atoms forming i-motif and G-quadruplex are stronger and stronger as the deterioration of the i-motif proceeds. Therefore, the work necessary to decompose the i-motif is higher and higher as the rmsd increases. In the original form of iG, as shown in the inset in Fig. 1, the i-motif is spatially well separated from the G-quadruplex and from the duplex, thus there are no extra interactions which enhance the stability of the i-motif. The unfolding of the G-quadruplex from the semi unfolded state (Fig. 8B) needs very similar amount of work like the unfolding from the original iG structure (Fig. 5). Thus, the presence of i-motif little affects the stability of the complementary G-quadruplex. However, at the neutral pH the difference between work plots seen in Fig. 8B may suggest some small stabilizing effect of the i-motif. Nevertheless, any G-quadruplex unfolding process (acidic or neutral pH) starting from any initial structure is difficult and cannot occur spontaneously. The i-motif unfolding is definitely easier at the neutral pH and the presence of the unfolded and disordered G-quadruplex forming fragment significantly enhances the stability of the i-motif at the neutral pH.