Visualization and Quantification of Single‐Base m6A Methylation

N⁶-methyladenosine (m⁶A) has emerged as the most prevalent form of RNA modification found across various RNA classes. The detection and quantification of m⁶A RNA modifications under various physiological conditions are crucial for elucidating disease mechanisms and identifying potential therapeutic targets. However, visualizing intracellular m⁶A 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 m⁶A RNA imaging. In this work, we introduce the TadA8.20-assisted N⁶-methyladenosine RNA imaging at single-base resolution (TARS) method for precise visualization and quantification of both A and m⁶A 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 m⁶A modifications at single-base resolution. TARS represents a novel tool that advances m⁶A RNA modification research by offering accurate and detailed insights into m⁶A modifications at the single-base level.