Detection of SARS-CoV‑2 RNA Using a DNA Aptamer Mimic of Green Fluorescent Protein

• Published in: ACS chemical biology 2022-04, Vol.17 (4), p.840-853
• Main Authors: VarnBuhler, Bria S., Moon, Jared, Dey, Sourav Kumar, Wu, Jiahui, Jaffrey, Samie R.
• Format: Article
• Language: English
• Subjects: Aptamers, Nucleotide - chemistry; COVID-19 - diagnosis; DNA - chemistry; Fluorescent Dyes - chemistry; Green Fluorescent Proteins; Humans; RNA - chemistry; RNA, Viral - genetics; SARS-CoV-2 - genetics
• ISSN: 1554-8929; 1554-8937
• DOI: 10.1021/acschembio.1c00893

RNA detection is important in diverse diagnostic and analytical applications. RNAs can be rapidly detected using molecular beacons, which fluoresce upon hybridizing to a target RNA but require oligonucleotides with complex fluorescent dye and quencher conjugations. Here, we describe a simplified method for rapid fluorescence detection of a target RNA using simple unmodified DNA oligonucleotides. To detect RNA, we developed Lettuce, a fluorogenic DNA aptamer that binds and activates the fluorescence of DFHBI-1T, an otherwise nonfluorescent molecule that resembles the chromophore found in green fluorescent protein. Lettuce was selected from a randomized DNA library based on binding to DFHBI-agarose. We further show that Lettuce can be split into two separate oligonucleotide components, which are nonfluorescent on their own but become fluorescent when their proximity is induced by a target RNA. We designed several pairs of split Lettuce fragments that contain an additional 15–20 nucleotides that are complementary to adjacent regions of the SARS-CoV-2 RNA, resulting in Lettuce fluorescence only in the presence of the viral RNA. Overall, these studies describe a simplified RNA detection approach using fully unmodified DNA oligonucleotides that reconstitute the Lettuce aptamer templated by RNA.