Development of an immobilized system for RNA modification analysis.

Characterizing RNA modifications is crucial for understanding fundamental biological processes, such as RNA folding, stability, translation, and splicing. However, current systems for ribonucleoside sample preparation are limited to the solution phase. In this study, we employed the click reaction between methyltetrazine and trans-cyclooctene to immobilize RNases, including nuclease P1, phosphodiesterase I, and shrimp alkaline phosphatase, on agarose beads. Using this digestion method, RNA was fully converted to ribonucleosides within 30 min. Importantly, integrating these immobilized RNases with a microspin tube modified with porous graphitic carbon enabled direct downstream MS analysis, constituting a streamlined system. We applied this system to monitor RNA modification dynamics during transforming growth factor-β (TGF-β)-induced epithelial-mesenchymal transition in lung cancer cells and observed significant changes in several RNA modifications (e.g. m6A and m5U), which is consistent with the indispensable role of RNA modifications in tumour metastasis. Overall, our results demonstrate the efficiency and robustness of our method and highlight a promising direction for RNA modification analysis, supporting the development of automated, high-throughput workflows for future large-cohort studies.