Direct Selection of Fluorescence-Enhancing RNA Aptamers

J Am Chem Soc. 2018 Mar 14;140(10):3583-3591. doi: 10.1021/jacs.7b10724. Epub 2018 Mar 5.

Abstract

RNA 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 affinity-a 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Aptamers, Nucleotide / chemistry*
  • Fluorescence*
  • Fluorescent Dyes / chemistry*
  • SELEX Aptamer Technique*

Substances

  • Aptamers, Nucleotide
  • Fluorescent Dyes