ja7b10724_si_001.pdf (991.89 kB)
Direct Selection of Fluorescence-Enhancing RNA Aptamers
Version 2 2018-03-05, 16:18
Version 1 2018-02-13, 01:13
journal contribution
posted on 2018-03-05, 16:18 authored by Michael Gotrik, Gurpreet Sekhon, Saumya Saurabh, Margaret Nakamoto, Michael Eisenstein, H. Tom SohRNA
aptamers that generate a strong fluorescence signal upon binding
a nonfluorescent small-molecule dye offer a powerful means for the
selective imaging of individual RNA species. Unfortunately, conventional
in vitro discovery methods are not efficient at generating such fluorescence-enhancing
aptamers, because they primarily exert selective pressure based on
target affinitya characteristic that correlates poorly with
fluorescence enhancement. Thus, only a handful of fluorescence-enhancing
aptamers have been reported to date. In this work, we describe a method
for converting DNA libraries into “gene-linked RNA aptamer
particles” (GRAPs) that each display ∼105 copies of a single RNA sequence alongside the DNA that encodes it.
We then screen large libraries of GRAPs in a high-throughput manner
using the FACS instrument based directly on their fluorescence-enhancing
properties. Using this strategy, we demonstrate the capability to
generate fluorescence-enhancing aptamers that produce a variety of
different emission wavelengths upon binding the dye of interest.