Structural dynamics of therapeutic nucleic acids with phosphorothioate backbone modifications.

Antisense oligonucleotides (ASOs) offer ground-breaking possibilities for selective pharmacological intervention for any gene product-related disease. Therapeutic ASOs contain extensive chemical modifications that improve stability to enzymatic cleavage and modulate binding affinity relative to natural RNA/DNA. Molecular dynamics (MD) simulation can provide valuable insights into how such modifications affect ASO conformational sampling and target binding. However, force field parameters for chemically modified nucleic acids (NAs) are still underdeveloped. To bridge this gap, we developed parameters to allow simulations of ASOs with the widely applied phosphorothioate (PS) backbone modification, and validated these in extensive all-atom MD simulations of relevant PS-modified NA systems representing B-DNA, RNA, and DNA/RNA hybrid duplex structures. Compared to the corresponding natural NAs, single PS substitutions had marginal effects on the ordered DNA/RNA duplex, whereas substantial effects of phosphorothioation were observed in single-stranded RNA and B-DNA, corroborated by the experimentally derived structure data. We find that PS-modified NAs shift between high and low twist states, which could affect target recognition and protein interactions for phosphorothioated oligonucleotides. Furthermore, conformational sampling was markedly altered in the PS-modified ssRNA system compared to that of the natural oligonucleotide, indicating sequence-dependent effects on conformational preference that may in turn influence duplex formation.