Detection of Large Genomic RNA via DNAzyme‐Mediated RNA Cleavage and Rolling Circle Amplification: SARS‐CoV‐2 as a Model
Jimmy Gu, Amal Mathai, Connor Nurmi, Dawn White, Gurpreet Panesar, Deborah Yamamura, Cynthia Balion, Jonathan B. Gubbay, Karen Mossman, Alfredo Capretta, Bruno J. Salena, Leyla Soleymani, Carlos D. M. Filipe, John D. Brennan, Yingfu Li
Abstract
A new method for the detection of genomic RNA combines RNA cleavage by the 10-23 DNAzyme and use of the cleavage fragments as primers to initiate rolling circle amplification (RCA). 230 different 10-23 DNAzyme variants were screened to identify those that target accessible RNA sites within the highly structured RNA transcripts of SARS-CoV-2. A total of 28 DNAzymes were identified with >20 % cleavage, 5 with >40 % cleavage and one with >60 % in 10 min. The cleavage fragments from these reactions were then screened for coupling to an RCA reaction, leading to the identification of several cleavage fragments that could efficiently initiate RCA. Using a newly developed quasi-exponential RCA method with a detection limit of 500 aM of RNA, 14 RT-PCR positive and 15 RT-PCR negative patient saliva samples were evaluated for SARS-CoV-2 genomic RNA, achieving a clinical sensitivity of 86 % and specificity of 100 % for detection of the virus in <2.5 h.