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.