DNAzyme-Enhanced CRISPR/Cas12a Cascade Enables Isothermal, One-Pot RNA Diagnostics

作者：Ruyue Wei, S Wang, Yufan Li, Na Li, Wei Pan, Bo Tang · 发表于：ACS Applied Materials & Interfaces · 年份：2026 · DOI：10.1021/acsami.6c05456 · 研究领域：Advanced biosensing and bioanalysis techniques、CRISPR and Genetic Engineering、Biosensors and Analytical Detection

While integrating DNAzymes with Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas systems offers a promising route to enhance CRISPR/Cas12a-based molecular diagnosis via enzyme-coupled cascade amplification, their implementation in simple, specific, and sensitive nucleic acid detection remains challenging, largely due to reliance on complex, multistep workflows. Here, we report an RNA-triggered DNAzyme circuit integrated with CRISPR/Cas12a that serves as a universal nucleic acid preamplifier, enabling one-pot and homogeneous detection. The catalytic activity of DNAzyme, initially suppressed by a complementary blocker strand, was restored upon the recognition of the target analyte. The activated DNAzyme then cleaved a hairpin-shaped substrate, liberating multiple activators that triggered a secondary CRISPR/Cas amplification reaction. This cascade generated a visible red band signal on a lateral flow assay via the collateral cleavage of a reporter. By employing the DNAzyme as a signal amplifier, the system efficiently converted a single RNA molecule into numerous initiators, breaking the one-to-one activation relationship between the target and Cas12a ribonucleoprotein and thereby greatly enhancing the detection sensitivity. Additionally, the system exhibited high programmability and universality, as a biosensor for a given target could be easily constructed by simply customizing the corresponding region of the blocker strand that is complementary to the ta...