Detection of SARS-CoV-2 RNA Using a DNA Aptamer Mimic of GreenFluorescent Protein

RNA detection is important in diverse diagnostic and analytical applications. RNAs can be rapidly detected usingmolecular beacons, whichfluoresce upon hybridizing to a target RNA but require oligonucleotides with complexfluorescent dye andquencher conjugations. Here, we describe a simplified method for rapidfluorescence detection of a target RNA using simpleunmodified DNA oligonucleotides. To detect RNA, we developed Lettuce, afluorogenic DNA aptamer that binds and activates thefluorescence of DFHBI-1T, an otherwise nonfluorescent molecule that resembles the chromophore found in greenfluorescentprotein. Lettuce was selected from a randomized DNA library based on binding to DFHBI-agarose. We further show that Lettucecan be split into two separate oligonucleotide components, which are nonfluorescent on their own but becomefluorescent whentheir proximity is induced by a target RNA. We designed several pairs of split Lettuce fragments that contain an additional 15-20nucleotides that are complementary to adjacent regions of the SARS-CoV-2 RNA, resulting in Lettucefluorescence only in thepresence of the viral RNA. Overall, these studies describe a simplified RNA detection approach using fully unmodified DNAoligonucleotides that reconstitute the Lettuce aptamer templated by RNA