Binding of antimiR or HDO-antimiR and miRNA from 20 Å away
At first, the distance between the antimiR or HDO-antimiR and the miRNA was fixed at 20 Å in initial state, and the angles were set in 8 directions at horizontal section of helix at 45° interval to consider the effect of initial configuration on the interaction (Figure 1, Supplementary Figures 1 and 2). Molecular simulations were performed 8 times (miR122) or 4 times (miR21) at each angle. We categorized the patterns of interaction between antimiR and miRNA as follows; Separated (no interaction), One-point interaction, Two-point interaction, Multiple-point interaction (more than two-point interaction).
As a result of simulation, all the four patterns were observed in both antimiR and HDO-antimiR (Figure 2). Then we investigated how the bonds occurred in patterns of Two-point or Multiple-point interaction. When a nucleic acid binds to a target one, hydrogen bonds are the main component, therefore we evaluated hydrogen bonds between the antimiR or HDO-antimiR and miRNA (Figure 3). From these results, there was no difference in the frequency of binding between antimiR and HDO-antimiR. So, we analyzed the number and location of hydrogen bonds formed between antimiR and HDO-antimiR about Two-point or more interaction pattern to investigate the mechanism of the difference in effect between antimiR and HDO-antimiR.
In the Two-point interaction pattern about antimiR with miRNA, hydrogen bonds were formed mainly through the atoms in the base site of the antimiR and miRNA near both ends, and no hydrogen bonds were observed in the phosphate site and the sugar site of antimiR. Similar to antimiR, hydrogen bond was mainly observed at the base site in the miRNA (Figure 4a, Supplementary Table 1). In the Multiple-point interaction pattern about antimiR with miRNA, which was observed only once, hydrogen bonds were formed at the middle part of antimiR and miRNA, and there was no selectivity about the binding site in the residues of antimiR. Similar to Two-point interaction pattern, hydrogen bonds were observed mainly at the base site of the miRNA (Figure 4b, Supplementary Tables 1 and 3). HDO-antimiR was bound to miRNA at multiple points from 3' end of antimiR-strand (5' end of cRNA-strand) to the middle part of HDO-antimiR in the Two-point and Multiple-point interaction pattern. Importantly, hydrogen bonds between HDO-antimiR and miRNA were formed through the atoms in the phosphate site and the sugar site of HDO-antimiR, and no hydrogen bond was observed in the base site of the HDO-antimiR. In HDO-antimiR, cRNA-strand was mainly bound to miRNA and there was no difference in binding frequency depending on the type of RNA. On the other hand, guanine bases mainly formed hydrogen bonds in miRNA (Figure 4c and 4d, Supplementary Tables 2 and 3).
While most of the hydrogen bonds of antimiR are formed via the nucleobase, those of HDO-antimiR are formed via atoms at the phosphate site except for the both ends of antimiR-strand and cRNA-strand. Then we evaluated the time course of the hydrogen bonds in the simulations about Two-point interaction pattern of antimiR and Multiple-point interaction pattern of HDO-antimiR. When the simulation was performed up to 5 nsec, the number of hydrogen bonds increased up to 4 (Two-point interaction pattern) or 6 (Multiple-point interaction pattern) in each case. In antimiR, the site where hydrogen bonds are formed is initially abundant at the 5' end of the antimiR, and then the hydrogen bonds further increase around the 5' end of the antimiR with the passage of time (Figure 5a). On the other hand, in HDO-antimiR, the site where hydrogen bonds are formed is initially abundant at the 5' end of the c-RNA strand and miRNA, and then the hydrogen bonds gradually increase toward the 3' end of the miRNA with the passage of time, and we observed miRNA entwined with HDO-antimiR (Figure 5b). During the simulation, most of the hydrogen bonds on the HDO-antimiR were phosphate site, and the atoms in the base site were not observed except for some of the both ends of antimiR-strand and cRNA-strand (Figure 5b).
Difference in binding site at the binding of antimiR or HDO-antimiR and miRNA
Next, in order to perform simulation from a state in which antimiR or HDO-antimiR and miRNA are closer to each other, nucleic acids were placed 5 Å away to the vertical axis direction (Figure 6, Supplementary Figures 3 and 4), then miRNA was placed in the major groove of HDO-antimiR shifted to -5 Å from miRNA (Figure 6), or minor groove of HDO-antimiR shifted to +5 Å from miRNA (Supplementary Figure 4). It is unlikely that miRNA will fit in the minor groove of HDO-antimiR, which is a narrow and shallow groove, so we considered the pattern that fits in the deep major groove. In the case of antimiR and miRNA, most hydrogen bonds were formed between the bases of each other as similar to the case of 20 Å separation on the axial section of helix (Figure 6a, Supplementary Tables 4 and 5). Like antimiR, HDO-antimiR and miRNA formed hydrogen bonds via the atoms at the base of HDO-antimiR, not the phosphate site of the nucleic acid, unlike when they were separated by 20 Å on the axial section of helix (Figure 6b, Supplementary Tables 4 and 5).
We further analyzed these results about the Two-point interaction pattern and Multiple-point interaction pattern when antimiR or HDO-antimiR is 5 Å shifted from miRNA in the vertical axis direction (both miR122 and miR21). At first, to find out which sites contribute to nucleic acid binding, we calculated the number of hydrogen bonds confirmed in this simulation for each binding site in the nucleic acid (Figure 7a-d and Supplementary Figure 5). In these results, there are several types of interaction patterns between HDO-antimiR or antimiR and miRNA. The atoms in the base part mainly form hydrogen bonds in antimiR, whereas in HDO-antimiR, the atoms in the phosphate bond site are mainly involved. On the miRNA side, the atoms at the base were mainly bound either to antimiR or HDO-antimiR. It was also found that the appearance rate of the Multiple-point interaction pattern, which seems to be the strongest binding form, differs depending on the initial conditions between HDO-antimiR and antimiR and miRNA.
In the time course about Multiple-point interaction pattern of HDO-antimiR and miRNA showed hydrogen bonds were not fixed, and the atom that once formed a hydrogen bond with another atom, then HDO-antimiR bound to the target miRNA gradually. By repeating these movements, it was expected that the miRNA would entwine in the major groove of HDO-antimiR. On the other hand, when HDO-antimiR and miRNA were placed 5 Å away to the vertical axis direction, miRNA was placed in the major groove of HDO-antimiR in the initial state. In the result of this simulation, HDO-antimiR and miRNA formed hydrogen bonds via the atoms at the base of HDO-antimiR, not the phosphate site of the nucleic acid.
Next, in order to investigate which bases affect the binding, we calculated the number of hydrogen bonds for each base in the nucleic acid, then it was corrected by dividing by the number of each base in the sequence in order to eliminate the variation in the number of bases since the number of bases differs depending on the sequence (Figure 7e-h and Supplementary Figure 6). In the results about shifted 5 Å in the vertical axis direction, antimiR showed the number of hydrogen bonds between guanine and cytosine is larger than them between thymine and adenine. These results partly can reflect the fact that the number of hydrogen bonds between guanine and cytosine is 3 and the number of hydrogen bonds between thymine and adenine is 2 when antimiR and miRNA form a complete double strand. On the other hand, there was no difference in the number of hydrogen bonds depending on the base in HDO-antimiR. Binding site analyses showed HDO-antimiR and miRNA formed hydrogen bonds via the atoms at the base of HDO-antimiR, not the phosphate site of the HDO-antimiR when HDO-antimiR and miRNA were placed 5 Å away to the vertical axis direction, and these data suggested HDO-antimiR is bound to miRNA via a base, but it is bound by a mechanism different from that of antimiR and miRNA. These results indicated it is not affected by the base sequence in HDO-antimiR because not base site, but phosphate site affects the bond in HDO-antimiR.