Characterization of the Intracellular Behavior of Mirror Image L-Oligonucleotides Reveals the Potent Cytotoxicity of G-Rich L-RNA

Due to their bioorthogonal properties, such as nuclease resistance, L-oligonucleotides are being increasingly employed in biomedical applications including L-aptamers, microarrays, molecular sensors, and live cell imaging probes. Given the large number of proteins that have been shown to bind nucleic acids in a nonspecific or “promiscuous” manner, it is reasonable to predict that interactions across the mirror are abundant, potentially leading to undesired effects. However how they interact with these environments and their potential consequences is not well studied. In this study, we conducted the first detailed characterization of how L-oligonucleotides interact with biological systems at the cellular level, revealing important stereochemistry-dependent behaviors of L-oligonucleotides. Notably, we show that G-rich L-oligonucleotides have potent cytotoxicity and a dramatic impact on gene expression, suggesting they make extensive interactions with endogenous proteins. In conclusion, the data presented here demonstrates that, similar to chemical modifications, stereochemistry can have a profound effect on the behavior of oligonucleotides in living systems. This work should motivate similar investigations into other modified oligonucleotides, such as XNAs, that are also perceived to be highly bioorthogonal, which may reveal unforeseen biological effects. Comparative gene expression of HeLa cell upon various L-RNA transfection.