Making target sites in large structured RNAs accessible to RNA-cleaving DNAzymes through hybridization with synthetic DNA oligonucleotides.

The 10-23 DNAzyme is one of the most active DNA-based enzymes, and in theory, can be designed to target any purine-pyrimidine junction within an RNA sequence for cleavage. However, purine-pyrimidine junctions within a large, structured RNA (lsRNA) molecule of biological origin are not always accessible to 10-23, negating its general utility as an RNA-cutting molecular scissor. Herein, we report a generalizable strategy that allows 10-23 to access any purine-pyrimidine junction within an lsRNA. Using three large SARS-CoV-2 mRNA sequences of 566, 584 and 831 nucleotides in length as model systems, we show that the use of antisense DNA oligonucleotides (ASOs) that target the upstream and downstream regions flanking the cleavage site can restore the activity (kobs) of previously poorly active 10-23 DNAzyme systems by up to 2000-fold. We corroborated these findings mechanistically using in-line probing to demonstrate that ASOs reduced 10-23 DNAzyme target site structure within the lsRNA substrates. This approach represents a simple, efficient, cost-effective, and generalizable way to improve the accessibility of 10-23 to a chosen target site within an lsRNA molecule, especially where direct access to the genomic RNA target is necessary.

Detection of Large Genomic RNA via DNAzyme-Mediated RNA Cleavage and Rolling Circle Amplification: SARS-CoV-2 as a Model.

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.