Mechanistic Insights into ASO-RNA Complexation: Advancing Antisense Oligonucleotide Design Strategies

Abstract

ABSTRACTOligonucleotide drugs, an emerging modulator class, hold promise for targeting previously undruggable biomacromolecules. To date, only 18 oligonucleotide drugs, including sought-after antisense oligonucleotides (ASO) and splice-switching oligonucleotides (SSO), have FDA approval. These agents effectively bind mRNA, inducing degradation or modulating splicing. Current oligonucleotide drug design strategies prioritize full Watson-Crick base pair complementarity, overlooking mRNA target 3D shapes. Given that mRNA conformational diversity can impact hybridization, incorporating mRNA key-structural properties into the design may expedite ASO lead discovery. Using atomistic molecular dynamics simulations inspired by experimental data, we demonstrate the advantages of incorporating common triple base pairs into the design of antisense oligonucleotides (ASOs) targeting RNA hairpin motifs, which are highly accessible regions for interactions. By employing an RNA pseudoknot modified into an ASO-hairpin complex, we investigate the effects of ASO length and hairpin loop mutations. Our findings suggest that ASO-mRNA complex stability is influenced by ASO length, number of common triple base pairs, and the dynamic accessibility of bases in the hairpin loop. Our study offers new mechanistic insights into ASO-mRNA complexation and underscores the value of pseudoknots in constructing training datasets for machine learning models aimed at designing novel ASO leads.