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.
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