A Programmable mRNA Platform for miRNA Detection via miRNA–mRNA ₂ Triplex-Mediated Ribosomal Frameshifting

Programmed −1 ribosomal frameshifting (−1 PRF) is a recoding mechanism utilized by viruses to expand their coding capacity and modulate the stoichiometric ratio of −1 frame and 0 frame translation products. The stability of mRNA secondary structure at the ribosomal entry site within the frameshifting stimulating elements (FSEs) determines the frameshifting efficiency. Here, we report the development of a programmable mRNA-based platform that detects specific mature microRNA (miRNA or miR) by converting their presence into a quantifiable protein output through miRNA-triggered −1 PRF. We designed a triplex-forming mRNA (TF-mRNA) platform to selectively trap target miRNAs through the formation of major-groove mRNA–miRNA–mRNA (miR–mRNA 2 ) triplexes. Biolayer interferometry and fluorescence binding studies confirmed that TF-mRNA forms stable complexes with cognate miRNAs with low nanomolar affinity and prolonged dissociation rate. Critically, the formation of miR–mRNA 2 triplex robustly stimulated ribosomal frameshifting in a cell-free dual-luciferase translation system, acting as a miRNA-dependent molecular switch. The generality of this TF-mRNA platform has been verified for several disease-associated purine-rich miRNAs, and it is suitable for targeting a wide range of other purine-enriched miRNAs. This programmable TF-mRNA platform establishes a foundation for developing novel diagnostic tools and synthetic biology circuits that convert the presence of miRNA into a quantifiable protein output.