RModBlock ASOs as a universal tool for precise and efficient inhibition of RNA modifications
The ability to selectively edit specific RNA modifications is crucial for understanding their roles in cellular function and disease. However, existing approaches, particularly dCas-based systems, have limited applicability, with performance that can vary depending on the modification type and target context. Here, we introduce the RNA Modification-Blocking (RModBlock) strategy, which employs chemically modified antisense oligonucleotides (ASOs) with locked nucleic acid to precisely inhibit modifications at targeted sites. Most RNA modification writers, including those for m5C and pseudouridine, require specific structural contexts. Using m5C as a representative modification with eraser proteins and pseudouridine as a representative modification without, we demonstrated that RModBlock ASOs efficiently blocked the formation of their structural context, inhibiting modified bases by up to 97% in human cells. Moreover, RModBlock ASOs also inhibited m6A, despite its writer protein not requiring a strict structural context, suggesting broad applicability. Unlike dCas13-eraser-based tools, which are limited to modifications with eraser proteins, the RModBlock strategy offers a versatile solution for targeting various modifications and achieves comparable or superior performance even when dCas13-eraser-based systems are applicable. RModBlock ASOs also show high specificity with minimal off-target effects. Finally, we highlight its therapeutic potential by inhibiting cancer-relevant modifications and achieving efficient in vivo delivery to the mouse liver via lipid nanoparticles. The RModBlock strategy offers a precise, efficient, and versatile platform for manipulating RNA modifications, with broad applicability in both basic and translational research.