Dual-Site Chemically Engineered MNAzymes for One-Pot Isothermal Detection of MicroRNAs.

Sensitive detection of microRNAs (miRNAs) remains challenging due to their low abundance and sequence similarity. Conventional DNAzyme- and multicomponent nucleic acid enzyme (MNAzyme)-based assays are enzyme-free but often limited by poor catalytic turnover. Here, we present a dual-site chemically engineered MNAzyme platform that integrates xeno-nucleic acid modification within the catalytic core and locked nucleic acid substitution within the target-binding arms to synergistically enhance catalytic and recognition efficiency. The resulting hybrid, termed LXMz, exhibits an ∼760-fold improvement in catalytic rate and an ∼450-fold increase in sensitivity relative to the unmodified MNAzyme. Under isothermal, one-pot conditions, LXMz achieves the quantitative detection of miR-92a with a limit of detection of 2 pM while maintaining single-base specificity. By combining catalytic enhancement and hybridization stabilization within a unified molecular design, this study establishes a new chemical engineering principle for constructing robust, enzyme-free biosensors. The LXMz framework provides a versatile and sensitive platform for miRNA analysis and holds promise for liquid biopsy and point-of-care diagnostics.