RNA modifications can affect RNase H1-mediated PS-ASO activity

• Published in: Molecular therapy. Nucleic acids 2022-06, Vol.28, p.814-828
• Main Authors: Doxtader Lacy, Katelyn A., Liang, Xue-hai, Zhang, Lingdi, Crooke, Stanley T.
• Format: Article
• Language: English
• Subjects: antisense oligonucleotide; cleavage; MT: Oligonucleotides: Therapies and Applications; Original; RNA modification; RNase H1; structure
• ISSN: 2162-2531; 2162-2531
• DOI: 10.1016/j.omtn.2022.05.024

Phosphorothioate modified antisense oligonucleotides (PS-ASOs) can reduce gene expression through hybridization to target RNAs and subsequent cleavage by RNase H1. Target reduction through this mechanism is influenced by numerous features of the RNA, which modulate PS-ASO binding affinities to the RNA target, and how the PS-ASO-RNA hybrid is recognized by RNase H1 for RNA cleavage. Endogenous RNAs are frequently chemically modified, which can regulate intra- and intermolecular interactions of the RNA. The effects of PS-ASO modifications on antisense activity have been well studied; however, much less is known regarding the effects of RNA modifications on PS-ASO hybridization and RNase H1 cleavage activity. Here, we determine the effects of three different RNA modifications on PS-ASO binding and antisense activity in recombinant and cell-based systems. Some RNA modifications can reduce PS-ASO hybridization, the cleavage activity of RNase H1, or both, while other modifications had minimal effects on PS-ASO function. In addition to these direct effects, RNA modifications can also change the RNA structure, which may affect PS-ASO accessibility in a cellular context. Our results elucidate the effects of three prevalent RNA modifications on PS-ASO-mediated RNase H1 cleavage activity, and such findings will help improve PS-ASO target site selection. [Display omitted] In this study, Doxtader Lacy and colleagues determine the effects of three prevalent RNA modifications on antisense activity via RNase H1 cleavage. The 2′-O-methyl and inosine modifications can significantly reduce PS-ASO-mediated RNA cleavage by RNase H1, while the m6A modification is more tolerated in the hybrid duplex.