Spatially resolved m⁶A profiling using m⁶A-ARTR-DBiT.

Abstract

N⁶-methyladenosine (m⁶A) on RNA plays diverse regulatory roles, yet its spatial distribution within tissues remains largely unexplored. Here we introduce m⁶A-ARTR-DBiT, a spatial m⁶A profiling assay that leverages reverse-transcription-based detection and deterministic barcoding in tissue to map transcriptome-wide m⁶A distribution while preserving native tissue context. Applying m⁶A-ARTR-DBiT to mouse embryonic tissues and adult brains generates spatially resolved m⁶A landscapes and reveals region-associated m⁶A features across different functional domains. Pairwise comparison of spatial m⁶A profiles with spatial transcriptomes uncovers positive correlations between m⁶A levels and the expression of its methyltransferases and binding proteins, which also enables systematic identification of tissue-region-specific epitranscriptomic regulation. In the mouse hippocampus, m⁶A-ARTR-DBiT allows for high-resolution mapping of m⁶A organization within fine-scale tissue structures. Together, m⁶A-ARTR-DBiT provides a platform for interrogating RNA modification distribution within intact tissue sections, offering insights into the link between spatially patterned m⁶A deposition and gene regulation.