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

ACS Chem Biol. 2022 Apr 15;17(4):840-853. doi: 10.1021/acschembio.1c00893. Epub 2022 Mar 26.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Aptamers, Nucleotide* / chemistry
  • COVID-19* / diagnosis
  • DNA / chemistry
  • Fluorescent Dyes / chemistry
  • Green Fluorescent Proteins
  • Humans
  • RNA / chemistry
  • RNA, Viral / genetics
  • SARS-CoV-2 / genetics

Substances

  • Aptamers, Nucleotide
  • Fluorescent Dyes
  • RNA, Viral
  • Green Fluorescent Proteins
  • RNA
  • DNA