四聚体中和与正交催化发夹组装用于microRNA直接差异检测方法开发

Abstract

Background MicroRNAs are valuable non-invasive biomarkers for breast cancer, yet their clinical utility in serial monitoring is limited by the need for absolute quantification and complex normalization in conventional detection methods. A platform capable of directly comparing miRNA abundance between two samples would simplify this process and reduce cumulative analytical errors. Results We present a proof-of-concept assay that directly compares miRNA levels through a tetrameric neutralization mechanism. Two hairpin probes, CNP1 and CNP2, capture target miRNAs to form dimeric complexes. When combined, equimolar dimers assemble into inert mCCm tetramers, while excess dimers from the sample with higher miRNA concentration trigger catalytic hairpin assembly with orthogonal SROMB probes, generating a binary, color-coded fluorescent readout (FAM for CNP1 excess, Texas Red for CNP2 excess) without requiring sample-level absolute quantification. Tetramer formation was confirmed by gel electrophoresis and solution-phase fluorescence quenching. The assay reliably detected concentration differences across varying ratios using four breast cancer subtype-associated miRNAs in spiked human serum and successfully accessed endogenous miRNA targets in total RNA extracted from MCF-7 cells. Semi-quantitative differential estimates showed 88.8% mean recovery, concordant with qRT-PCR. Limits of detection determined from fluorescence-versus-concentration standard curves ranged from 3 to 64 pM across SROMB detection channels, and four-parameter logistic calibration enabled semi-quantitative estimation of differential changes. Significance This platform provides a methodological foundation for direct differential miRNA detection by executing sample comparison at the molecular level. While current sensitivity remains above typical circulating miRNA concentrations and requires further optimization, this approach, following rigorous clinical validation, may facilitate comparative biomarker a