Visualization and Quantification of Single‐Base m⁶A Methylation

Abstract N 6 ‐methyladenosine (m 6 A) has emerged as the most prevalent form of RNA modification found across various RNA classes. The detection and quantification of m 6 A RNA modifications under various physiological conditions are crucial for elucidating disease mechanisms and identifying potential therapeutic targets. However, visualizing intracellular m 6 A modifications at single‐base resolution remains a significant challenge. Existing methods based on high‐throughput sequencing or in vitro assays are not suitable for in situ m 6 A RNA imaging. In this work, we introduce the TadA8.20‐assisted N 6 ‐methyladenosine RNA imaging at single‐base resolution (TARS) method for precise visualization and quantification of both A and m 6 A forms at specific RNA sites within single cells. Validation studies using TARS on MALAT1 lncRNA in HeLa cells and CCND1 mRNA in breast cancer cell lines demonstrated its high specificity and efficiency in mapping and quantifying m 6 A modifications at single‐base resolution. TARS represents a novel tool that advances m 6 A RNA modification research by offering accurate and detailed insights into m 6 A modifications at the single‐base level.