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<p>Site-specific detection of N⁶-methyladenosine (m<sup>6</sup>A) 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 m<sup>6</sup>A detection. The strategy exploits m<sup>6</sup>A-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&#xa0;nM, with high selectivity against unmodified and non-complementary RNAs. Applied to human serum, the method quantifies site-specific m<sup>6</sup>A on the lncRNA MALAT1 and successfully distinguishes osteosarcoma patients from healthy controls. This work presents a practical approach for site-resolved m<sup>6</sup>A analysis without nucleic acid preamplification.</p> Graphical Abstract <p></p>

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

  • 期刊论文

  • 发表日期：

    2026年3月26日

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  • Weifeng Zhang,
  • Xiangming Li,
  • Huihui Shi,
  • Jing Liang,
  • Xiao Ma,
  • Xiaoyan Xu,
  • Chuqi Zhang,
  • Mingyue Xiong

摘要

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.

Graphical Abstract