期刊
NUCLEIC ACIDS RESEARCH
卷 44, 期 6, 页码 2491-2500出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkw083
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资金
- LABEX [ANR-10-LABX-0036_NETRNA]
- French National Research Agency as part of the Investments for the future program
- Universite de Strasbourg
- Centre National de la Recherche Scientifique
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 (k(cat)) of a model ribozyme.
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