Strand displacement-driven dual-nuclease cascade strategy for preamplification-free and site-specific RNA m⁶A...

Site-specific detection of N⁶-methyladenosine (m⁶A) in RNA remains challenging due to its lack of distinctive chemical reactivity and preservation of canonical base pairing. Here, we report a strand displacement-driven dual-nuclease cascade (SDD) for preamplification-free, site-specific m⁶A detection. The strategy exploits m⁶A-dependent resistance to MazF cleavage at ACA sites, enabling intact target RNA to trigger toehold-mediated strand displacement and form a DSN-cleavable DNA-RNA hybrid. This hybrid activates DSN-mediated target recycling, leading to iterative release of a Cas13a activator strand and amplified fluorescence readout. The SDD assay achieves a limit of detection of 4.24 fM over a linear dynamic range from 1 fM to 10 nM, with high selectivity against unmodified and non-complementary RNAs. Applied to human serum, the method quantifies site-specific m⁶A on the lncRNA MALAT1 and successfully distinguishes osteosarcoma patients from healthy controls. This work presents a practical approach for site-resolved m⁶A analysis without nucleic acid preamplification.