Authors: Laura V. Croft  Mark Fisher, Tabassum Khair Barbhuiya, Serene El-Kamand, Samuel Beard, Aleksandra Rajapakse, Roland Gamsjaeger, Liza Cubeddu, Emma Bolderson, Ken O’Byrne , Derek Richard derek.richard@qut.edu.au, and Neha S. Gandhi

Published Online: 17 June 2024

Abstract

Single-stranded oligonucleotides (SSOs) are a rapidly expanding class of therapeutics that comprises antisense oligonucleotides, microRNAs, and aptamers, with ten clinically approved molecules. Chemical modifications such as the phosphorothioate backbone and the 2′-O-methyl ribose can improve the stability and pharmacokinetic properties of therapeutic SSOs, but they can also lead to toxicity in vitro and in vivo through nonspecific interactions with cellular proteins, gene expression changes, disturbed RNA processing, and changes in nuclear structures and protein distribution. In this study, we screened a mini library of 277 phosphorothioate and 2′-O-methyl-modified SSOs, with or without mRNA complementarity, for cytotoxic properties in two cancer cell lines. Using circular dichroism, nucleic magnetic resonance, and molecular dynamics simulations, we show that phosphorothioate- and 2′-O-methyl-modified SSOs that form stable hairpin structures through Watson–Crick base pairing are more likely to be cytotoxic than those that exist in an extended conformation. In addition, moderate and highly cytotoxic SSOs in our dataset have a higher mean purine composition than pyrimidine. Overall, our study demonstrates a structure–cytotoxicity relationship and indicates that the formation of stable hairpins should be a consideration when designing SSOs toward optimal therapeutic profiles.

Introduction
Over the past three decades, nucleic acid-based therapies have become a vital drug discovery platform alongside small molecules and antibodies. Nucleic acid therapies encompass single-stranded oligonucleotide (SSO) therapeutics such as antisense oligonucleotides (ASOs), microRNAs, and aptamers; double-stranded oligonucleotides such as short interfering RNAs (siRNAs); and messenger RNAs (mRNAs).¹ Eighteen nucleic acid therapeutics have been approved by the U.S. Food and Drug Administration (FDA) and/or European Medicines Agency (EMA), including nine ASOs, five siRNAs, one aptamer, one vaccine adjuvant, and two mRNA vaccines.^1–4 In addition, around 30 experimental nucleic acid drugs are in late-stage clinical development for indications such as cancer, amyloidosis, nephropathy, ischemic heart disease, cystic fibrosis, wound healing, and others.¹

Continue reading here