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

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'-<i>O</i>-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.