Programmable Split DNAzyme Modulators via Allosteric Cooperative Activation for mRNA Electrochemiluminescence Biosensing

作者：Liu-Qing Tan, Weijia Zeng, Qiaolin Chen, Xiu‐Li Tao, Ruo Yuan, Yan‐Mei Lei, Ying Zhuo · 发表于：Analytical Chemistry · 年份：2025 · DOI：10.1021/acs.analchem.4c07071 · 被引用次数：12 · 研究领域：Advanced biosensing and bioanalysis techniques、RNA and protein synthesis mechanisms、Biosensors and Analytical Detection

DNAzymes, known for their programmability, stability, and cost-effectiveness, are powerful tools for signal transduction in complex biological systems. However, their application in responding to target effectors is often hindered by limited catalytic efficiency and susceptibility to unintended activation. Here we propose an allo s teric coopera t ive a ctivation strategy to program a spli t DNAzym e modulato r (STATER) that enables sensitive and accurate electrochemiluminescence (ECL) biosensing of interleukin-6 (IL-6) mRNA. Our design features a STATER that leverages a DNA tetrahedron as a central scaffold, equipped with two pairs of T-shaped hairpin probes (TP) and helper hairpin probes (HP). Specifically, the TP contains two apurinic/apyrimidinic endonuclease 1 (APE1) recognition sites, an IL-6 mRNA recognition region, and a partzyme fragment, while the HP contains a corresponding paired partzyme fragment. Unlike conventional DNAzyme modulators that rely on single effector activation, the STATER integrates an allosteric cooperative activation mechanism, which ensures that all preblocked components are synergistically activated and assembled within a confined space, facilitating rapid and specific reconstruction of the DNAzyme’s catalytic active domain. Furthermore, upon cooperative recognition by APE1 and IL-6 mRNA, two inactive partzymes undergo an allosteric assembly via a toehold exchange displacement reaction, switching on the cleavage reactivity of STATER. This mecha...