モジュール式RNAアプタマーバイオセンサーの構造ガイドエンジニアリングによる、ワンポット・マルチプレックスcircRNA検出

Circular RNAs (circRNAs) are a class of endogenous biological macromolecules characterized by their covalently closed, single-stranded structures, which confer exceptional stability and sequence-specific back-splice junctions (BSJs). Exploiting these unique structural features for diagnostic purposes remains challenging, particularly in multiplexed formats. Here, we report a modular biosensing platform that integrates the structural specificity of circRNA BSJ recognition with the conformation-dependent fluorescence emission of engineered RNA aptamers. This system, termed FRA-MOML, utilizes ligase-assisted assembly to convert target recognition into a transcribable DNA template, which is then amplified by T7 RNA polymerase to produce structural switches-fluorescent RNA aptamers (e.g., iSpinach and Mango). These aptamers, upon folding into their specific three-dimensional structures, bind to small-molecule fluorogens and emit distinct fluorescent signals. By decoupling the structural recognition module (probes) from the signal-generating macromolecule (aptamer), our platform achieves femtomolar sensitivity, excellent single-nucleotide discrimination (especially at structurally critical ligation junctions), and one-pot multiplex detection without labeled probes. We validated this structure-based design by simultaneously quantifying two bladder cancer-associated circRNAs (circSMARCA5 and circSLC38A1) in complex biological matrices, including serum, cell lines, and tumor tissues. The plug-and-play programmability, rooted in the modular architecture of nucleic acids, offers a versatile tool not only for circRNA detection but also for studying structure-function relationships of diverse biological macromolecules.