iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications

Nucleic Acids Res. 2016 Apr 7;44(6):2491-500. doi: 10.1093/nar/gkw083. Epub 2016 Mar 1.

Abstract

Using random mutagenesis and high throughput screening by microfluidic-assisted In Vitro Compartmentalization, we report the isolation of an order of magnitude times brighter mutants of the light-up RNA aptamers Spinach that are far less salt-sensitive and with a much higher thermal stability than the parent molecule. Further engineering gave iSpinach, a molecule with folding and fluorescence properties surpassing those of all currently known aptamer based on the fluorogenic co-factor 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). We illustrate the potential of iSpinach in a new sensitive and high throughput-compatible fluorogenic assay that measures co-transcriptionally the catalytic constant (kcat) of a model ribozyme.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aptamers, Nucleotide / chemical synthesis
  • Aptamers, Nucleotide / chemistry*
  • Base Sequence
  • Benzyl Compounds / chemistry*
  • Biocatalysis
  • Fluorescent Dyes / chemical synthesis
  • Fluorescent Dyes / chemistry*
  • Gene Library
  • High-Throughput Screening Assays*
  • Humans
  • Imidazolines / chemistry*
  • Kinetics
  • Microfluidics / instrumentation
  • Microfluidics / methods
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Polymerase Chain Reaction / methods
  • RNA, Catalytic / chemistry*
  • RNA, Catalytic / genetics
  • RNA, Catalytic / metabolism
  • SELEX Aptamer Technique
  • Transcription, Genetic

Substances

  • 3,5-difluoro-4-hydroxybenzylidene imidazolinone
  • Aptamers, Nucleotide
  • Benzyl Compounds
  • Fluorescent Dyes
  • Imidazolines
  • RNA, Catalytic