Enhancing Clinical Detection Accuracy of Large Structured Viral RNA via DNAzyme Cleavage and Antisense‐Assisted Rolling Circle Amplification

Abstract Sensitive detection of viral RNA is critical for accurate diagnostic testing, particularly during outbreaks of emerging infectious diseases. Rolling circle amplification (RCA) is a powerful isothermal amplification strategy that can be directly primed by RNA, eliminating the need for reverse transcription. Previous approaches have used 10–23 DNAzymes to cleave viral RNA, generating 3′‐ends for hybridization to circular DNA templates (CDTs). However, the resulting RNA fragments often retained secondary or tertiary structures that hindered CDT binding and limited RCA efficiency. To address this challenge, we developed antisense oligonucleotide‐assisted RCA (ASO‐RCA), a general strategy that uses short upstream antisense oligonucleotides (ASOs) to remodel RNA structure and expose the CDT‐binding site. Using five DNAzyme‐CDT systems targeting distinct regions of the SARS‐CoV‐2 genome, we show that ASO inclusion improves CDT hybridization and enhances RCA output—by up to 70‐fold. This enhancement was observed using both linear and quasi‐exponential RCA formats and remained effective in 50% pooled saliva. When applied to clinical saliva samples, ASO‐assisted RCA markedly improved diagnostic performance, achieving 100% sensitivity and up to 97.5%–100% accuracy across multiple systems. These findings establish ASO‐DNAzyme‐RCA as a simple, robust, and clinically relevant platform for improving nucleic acid detection in structured RNA targets.