(A) Schematic of PACRAT. Reactions begin with an RNA target and reverse transcriptase reverse transcribes the single-stranded RNA to cDNA. DNA polymerase completes synthesis of a complementary strand, making a double-stranded DNA template. RPA begins with recombinases binding single-stranded primers, which then invade dsDNA. SSBs bind single-stranded DNA to stabilize it and DNA polymerase synthesizes a new DNA strand in each direction. DNA synthesis continues until two new DNA duplexes are formed. The dsDNA products have a T7 RNA polymerase promoter region (blue) and an aptamer coding sequence (green). T7 RNA polymerase can then transcribe the dsDNA product, resulting in an RNA amplicon conjugated to a fluorescent aptamer. The aptamer can bind its small-molecule cognate dye, generating a specific, real-time fluorescence signal. (B) Initial validation of PACRAT with urea-PAGE gels. SYBR Gold stained urea-PAGE shows the size of the DNA amplicon produced by RPA amplification at 39°C. The lane containing the reaction with 4.5 × 1010 copies of SARS-CoV-2 nucleocapsid N3 gene target shows the correct band size for the product at 155 bp. (C) Urea-PAGE gels of RNA products from PACRAT reactions with SARS-CoV-2 versus reactions with no target. (Left) SYBR-stained gel shows the correct RNA product size of 127 nt. (Right) DFHBI-stained gel, in which DFHBI is the cognate dye for the Broccoli aptamer, shows the band that contains the Broccoli aptamer.
