Antibody-Free and Highly Sensitive Detection of Site-Specific m⁶A Modification via Terminal-Mediated m⁶A PCR

N6-Methyladenosine (m⁶A) is the most prevalent RNA chemical modification in epitranscriptomics and is closely associated with disease development. Current m⁶A detection techniques predominantly rely on specific antibodies to capture modified transcripts, followed by detection through sequencing or PCR. However, these methods are complex to operate and often suffer from limited resolution, as well as low sensitivity. Here, we developed a unique site-specific m⁶A detection strategy denoted as STEM-M6A-PCR (Specific Terminal-Mediated m⁶A PCR). This approach employs a well-designed capture primer and the specific properties of Bst DNA polymerase to trigger the generation of a specific end that forms a complete hairpin structure, enabling exponential amplification for highly sensitive detection. STEM-M6A-PCR achieves a detection limit of 100 aM for target m⁶A-RNA with a total RNA input as low as 250 fg. Notably, this method is extraordinarily capable of site-specific resolution for closely adjacent m⁶A modifications through the rational design of capture primers. As an antibody-independent approach, STEM-M6A-PCR offers a powerful tool for the precise identification and quantification of m⁶A modifications in biological studies.