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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\u201323 DNAzymes to cleave viral RNA, generating 3\u2032\u2010ends 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\u2010assisted RCA (ASO\u2010RCA), a general strategy that uses short upstream antisense oligonucleotides (ASOs) to remodel RNA structure and expose the CDT\u2010binding site. Using five DNAzyme\u2010CDT systems targeting distinct regions of the SARS\u2010CoV\u20102 genome, we show that ASO inclusion improves CDT hybridization and enhances RCA output\u2014by up to 70\u2010fold. This enhancement was observed using both linear and quasi\u2010exponential RCA formats and remained effective in 50% pooled saliva. When applied to clinical saliva samples, ASO\u2010assisted RCA markedly improved diagnostic performance, achieving 100% sensitivity and up to 97.5%\u2013100% accuracy across multiple systems. These findings establish ASO\u2010DNAzyme\u2010RCA as a simple, robust, and clinically relevant platform for improving nucleic acid detection in structured RNA targets.<\/jats:p>","DOI":"10.1002\/anie.202507973","type":"journal-article","created":{"date-parts":[[2025,6,17]],"date-time":"2025-06-17T15:13:22Z","timestamp":1750173202000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Enhancing Clinical Detection Accuracy of Large Structured Viral RNA via DNAzyme Cleavage and Antisense\u2010Assisted Rolling Circle Amplification"],"prefix":"10.1002","volume":"64","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7066-9672","authenticated-orcid":false,"given":"Amal","family":"Mathai","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Biomedical Sciences McMaster University  1280 Main Street West Hamilton ON L8S 4K1 Canada"},{"name":"Biointerfaces Institute McMaster University  1280 Main Street West Hamilton ON L8S 4M1 Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jimmy","family":"Gu","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biomedical Sciences McMaster University  1280 Main Street West Hamilton ON L8S 4K1 Canada"},{"name":"Biointerfaces Institute McMaster University  1280 Main Street West Hamilton ON L8S 4M1 Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Connor","family":"Nurmi","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biomedical Sciences McMaster University  1280 Main Street West Hamilton ON L8S 4K1 Canada"},{"name":"Biointerfaces Institute McMaster University  1280 Main Street West Hamilton ON L8S 4M1 Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3461-9824","authenticated-orcid":false,"given":"John D.","family":"Brennan","sequence":"additional","affiliation":[{"name":"Biointerfaces Institute McMaster University  1280 Main Street West Hamilton ON L8S 4M1 Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7533-6743","authenticated-orcid":false,"given":"Yingfu","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biomedical Sciences McMaster University  1280 Main Street West Hamilton ON L8S 4K1 Canada"},{"name":"Biointerfaces Institute McMaster University  1280 Main Street West Hamilton ON L8S 4M1 Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,6,26]]},"reference":[{"key":"e_1_2_9_1_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cca.2024.117792"},{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1002\/jbio.202400243"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10544-022-00631-1"},{"key":"e_1_2_9_4_1","first-page":"1194","volume":"24","author":"Senkus E.","year":"2019","journal-title":"Ann. 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