3.1 Most Stable Conformation and allowed flexibility of each modification:
All the conformations generated from the TANGO tool were sorted based on the PM6 Hamiltonion energies. The start structure and the most stable conformation of each modification are shown in Figure 2a and 2b. The most stable conformation which had the lowest energy compared to the other conformations was identified and all the torsion angles were measured for the same. Marking of torsion angles (α, β, γ, δ, ε, andχ ) are shown in Figure 3. The PM6 energies and the torsion angles of the most stable conformations for all the selected antisense modifications are given in Table 2. The glycosidic torsion angle of all the modifications has been found to favour the anti conformation except for the RCS and CMS modifications. The number of torsion angles rotated, the number of conformations generated for each modification and the number of conformations found within the range of 10 Kcal/mol from the lowest conformation of respective modifications are given in Supplementary Table S1.
The ensembles of conformations having PM6 energy below 10 Kcal/mol were plotted by considering the energy and RMSD as shown in Figure 4. This plot captures the stable regions and flexibility allowed with each modification. RMSD values explain the structural similarity and differences. A lower RMSD value suggests more structural similarity and vice versa But, most of the structures/ clusters were below 2 Å. It was observed that the highly populated cluster was not close to the lowest energy for the majority of modifications. Some modifications have not even shown any highly populated clusters. CME modification has shown the highest population of clusters among all the modifications due to the presence of an extra 2-O methoxyethyl free chain and thus a huge number of conformations were generated having energy within 10 Kcal/mol from the lowest energy conformation.
The antisense modifications chosen from literature like the DCS showed higher values of energy whereas CME showed lower values of energy in their respective clusters. The modification A1 & A5 were exhibiting populated clusters but A2, A3, and A4 exhibiting restricted flexibility of the modifications. It was also observed from the sugar puckering of each modification that the DC and DCS were showing C2’ endo puckering which is a property of B-form helix generally observed in DNA. The modifications RC, RCS, LCC, LCS, CME, and CMS were showing C3’ endo puckering, a property of A-form helix in nucleic acids. All the proposed novel modifications namely A1, A2, A3, A4 and A5 were seen to prefer C3’ endo puckering.