Interrogation of mirror-image l-RNA–protein interactions reveals key mechanisms of single-stranded G-rich l-RNA cytotoxicity and a potential mitigation strategy

Abstract

L-Oligonucleotides (ONs), the synthetic enantiomers of native D-nucleic acids, are being increasingly utilized in the development of diverse biomedical technologies, including molecular imaging tools, diagnostic biosensors, and aptamer-based therapeutics. Nevertheless, our understanding of how L-ONs behave in living systems falls far short of native D-ONs. In particular, despite the potential for an abundant L-ON-protein interactome, the extent to which L-ONs bind to endogenous proteins and the consequences of these interactions are unknown, posing a major hurdle towards engineering functional L-ONs with predictable intracellular behaviours. Towards closing this knowledge gap, we now report the first L-ON–protein interactome, revealing that a wide-range of nuclear proteins have the potential to bind L-RNA. Importantly, by focusing our study on cytotoxic single-stranded G-rich L-RNA sequences, our data reveal key protein interactions that contribute to the cytotoxicity of these sequences. Furthermore, we show that introducing 2′-O-methyl modifications into single-stranded G-rich L-RNA can decrease its cytotoxicity through reducing L-RNA–protein interactions, thereby demonstrating that a well-established strategy for mitigating the cytotoxic effects of antisense ONs may translate across the chiral mirror. Overall, these findings greatly deepen our understanding of the intracellular behavior of L-ONs and provide valuable guidance for the future development of safe and effective L-ON-based biomedical technologies.