Transforming RNA Degradation into Accurate RNA Detection: RNase I–Assisted Rolling Circle Amplification for Clinical Samples

Abstract Ribonucleases are powerful degradative enzymes that rapidly cleave RNA and have long been regarded as obstacles to RNA diagnostics. Detecting large, structured RNA molecules in biological samples is further complicated by extensive secondary structures and abundant background RNA. Here we transform this destructive activity into a diagnostic advantage through a RNase I–assisted rolling circle amplification (RI‐RCA) strategy that integrates enzymatic RNA digestion with circular DNA templates (CDTs) to achieve direct, reverse‐transcription‐free RNA detection. In this system, Escherichia coli RNase I—a widely available endoribonuclease—selectively digests structured RNA to expose complementary CDT‐binding sites, which are immediately protected through duplex formation and converted into primers for RCA. The resulting reaction proceeds isothermally at room temperature in linear, quasi‐exponential, and exponential modes, achieving quantitative signal generation across concentrations from 50 aM to 50 fM and maintaining performance in 50% pooled human saliva. Applied to clinical saliva samples from SARS‐CoV‐2–positive and –negative individuals, RI‐RCA achieved near‐perfect sensitivity and 98–100% diagnostic accuracy. By coupling enzymatic RNA digestion with sequence‐specific protection via CDT hybridization, this strategy converts RNA instability from a liability into an analytical advantage, providing a simple, robust, and clinically translatable platform for accurate detection of structured RNA targets.