A Real-Time, Programmable, and Multiplex Ligation Chain Reaction-MNAzyme Platform for Single-Nucleotide Variant Detection.

Here, we present a modular and programmable real-time quantitative detection platform that integrates ligase chain reaction (LCR) with multicomponent nucleic acid enzymes (MNAzymes) for single-nucleotide variant (SNV) analysis. By incorporating a universal MNAzyme-based reporter, the LCR-MNAzyme system enables PCR-like real-time quantitative monitoring of SNVs while supporting programmable and multiplex detection. The target recognition module allows precise quantification of multiple variants, while reconfiguring the signal output module enables multiplex detection in a single homogeneous reaction. To further enhance analytical performance, glyoxal-caged probes were incorporated to achieve hot-start LCR, effectively suppressing background amplification and improving detection sensitivity. The optimized system exhibits a linear range from 10 aM to 10 pM with a detection limit of 5.79 aM and enables quantification of the JAK2 V617F mutation down to a variant allele frequency of 0.03%. Quantitative results from clinical samples showed excellent agreement with sequencing data. This work establishes a generalizable framework for highly sensitive, specific, and multiplex SNV detection, advancing the development of quantitative nucleic acid analysis tools for precision molecular research.