Nonenzymatic detection and imaging of m6A-methylated RNA via proximity hybridization-initiated hybridization chain reaction with site-specific recognition.

Abstract

N⁶-methyladenosine (m6A) is a critical epitranscriptomic modification involved in diseases such as liver fibrosis and cancer. Imaging site-specific m6A methylation in intact cells with high resolution remains challenging, as current methods typically rely on enzymatic ligation or primer exchange amplification, which require external triggers and temperature-sensitive enzymes. In the current study, we present an enzyme-free amplification strategy, proximity hybridization-initiated hybridization chain reaction (PH-HCR), for in situ visualization of m6A-methylated RNAs. Dual probes recognizing both the target RNA sequence and the m6A modification enable proximity-dependent initiation of an autonomous HCR cascade without any exogenous initiator or enzymatic catalysis, generating fluorescent DNA nanowires for amplified signal generation. PH-HCR achieves a detection limit of 1.22 pM for synthetic m6A-containing transforming growth factor (TGF)-β1 mRNA, enables visualization of m6A-associated RNA changes in living cells, and reveals cell-to-cell heterogeneity of m6A-associated signals at single-cell resolution. The method is applicable to different RNA targets and cell types. By eliminating the need for ligases, polymerases, or external triggers, PH-HCR offers a simple, robust, and biocompatible tool for studying the spatiotemporal dynamics of m6A-modified RNAs in basic research and diagnostics.