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ISpinach: a fluorogenic RNA aptamer optimized for in vitro applications.

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.

SUBMITTER: Autour A

PROVIDER: S-EPMC4824111 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications.

Autour Alexis A Westhof Eric E Ryckelynck Michael M

Nucleic acids research 20160301 6

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-difluo ...[more]

PMID: 26932363

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