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bioRxiv · 10.1101/2025.11.24.690164

A programmable mRNA platform for miRNA detection via miRNA-mRNA2 triplex-mediated ribosomal frameshifting

Chen, Y.·Zhao, W.·Chen, H.·Zhou, H.·Fan, S.·Duan, H.·Dai, Y.·Lu, R.·Li, C.·Jiang, C.·Chan, E.·Chen, G.
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Abstract

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-mRNA2) triplexes. Bio-layer 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-mRNA2 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. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/690164v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1d6d9f7org.highwire.dtl.DTLVardef@8008e6org.highwire.dtl.DTLVardef@1c8344borg.highwire.dtl.DTLVardef@e153ec_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Chen, Y., Zhao, W., Chen, H., Zhou, H., Fan, S., Duan, H., Dai, Y., Lu, R., Li, C., Jiang, C., Chan, E., Chen, G.. 2025-11-24. A programmable mRNA platform for miRNA detection via miRNA-mRNA2 triplex-mediated ribosomal frameshifting. https://doi.org/10.1101/2025.11.24.690164

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