Enzyme-free and isothermal discrimination of microRNA point mutations using a DNA split proximity circuit with turn-on fluorescence readout

MicroRNAs (miRNAs) hold immense potential as disease biomarkers, yet their short lengths and high sequence homology pose unique challenges in detection. Conventional methods such as the gold standard qRT-PCR and other isothermal amplification methods require sophisticated primer designs and use of enzymes which add uncertainties to the assay robustness. In this work, we demonstrate the use of a plug-and-play molecular detection platform, termed split proximity circuit (SPC), to achieve a selectivity comparable to qRT-PCR in differentiating point mutations using several miRNAs as proof-of-concept models. The analytical sensitivity of SPC has been improved by a hundred-fold over our previous work and matches/outperforms the enzyme-free assays reported in the literature by evolving the core signal-generating domains. Key design changes include improved hybridization chain reaction (HCR) hairpin sequences and the incorporation of a turn-on fluorescence signal based on fluorophore-quencher format. The core domains were then kept constant while redesigning the target recognition region to be complementary to various target sequences, all of which yield similar analytical performance. Notably, SPC maintained robust signal recovery with low variance even in complex biological matrices. With its enzyme-free and single room temperature operation, SPC presents a promising platform for quick and easy miRNA quantification.