Programmable Split DNAzyme Modulators via AllostericCooperative Activation for mRNA Electrochemiluminescence Biosensing

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<b><u>s</u></b>teric coopera<b><u>t</u></b>ive <b><u>a</u></b>ctivation strategy to program a spli<b><u>t</u></b> DNAzym<b><u>e</u></b> modulato<b><u>r</u></b> (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 mechanism enables the establishment of an activation threshold for IL-6 mRNA, thereby minimizing nonspecific activation in complex scenarios. Our studies demonstrate that the STATER exhibits outstanding sensitivity and selectivity for IL-6 mRNA detection using the supramolecular gold nanoclusters network-based ECL platform. The biosensor provides a linear detection span from 1 × 10^–13 to 1 × 10^–7 M, with a limit of detection as low as 3.26 × 10^–14 M, highlighting STATER’s potential for detecting various analytes in complex biological systems.