Journal Article

iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications

Alexis Autour, Eric Westhof and Michael Ryckelynck

in Nucleic Acids Research

Volume 44, issue 6, pages 2491-2500
Published in print April 2016 | ISSN: 0305-1048
Published online March 2016 | e-ISSN: 1362-4962 | DOI: https://dx.doi.org/10.1093/nar/gkw083

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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.

Journal Article.  7251 words.  Illustrated.

Subjects: Research Methods in Life Sciences

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