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

TitleDetection of SARS-CoV-2 RNA Using a DNA Aptamer Mimic of Green Fluorescent Protein.
Publication TypeJournal Article
Year of Publication2022
AuthorsVarnBuhler BS, Moon J, Dey SKumar, Wu J, Jaffrey SR
JournalACS Chem Biol
Volume17
Issue4
Pagination840-853
Date Published2022 Apr 15
ISSN1554-8937
KeywordsAptamers, Nucleotide, COVID-19, DNA, Fluorescent Dyes, Green Fluorescent Proteins, Humans, RNA, RNA, Viral, SARS-CoV-2
Abstract

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.

DOI10.1021/acschembio.1c00893
Alternate JournalACS Chem Biol
PubMed ID35341244
Grant ListR35 NS111631 / NS / NINDS NIH HHS / United States

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