title: 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.
authors.affiliation: Architecture et Réactivité de l'ARN, Université de Strasbourg, Institut de biologie moléculaire et cellulaire du CNRS, 15 rue René Descartes, 67084, Strasbourg, France.
authors.forename: Alexis
authors.initials: A
authors.lastname: Autour
authors.affiliation: Architecture et Réactivité de l'ARN, Université de Strasbourg, Institut de biologie moléculaire et cellulaire du CNRS, 15 rue René Descartes, 67084, Strasbourg, France.
authors.forename: Eric
authors.identifier: http://orcid.org/0000-0002-6172-5422
authors.initials: E
authors.lastname: Westhof
authors.affiliation: Architecture et Réactivité de l'ARN, Université de Strasbourg, Institut de biologie moléculaire et cellulaire du CNRS, 15 rue René Descartes, 67084, Strasbourg, France m.ryckelynck@ibmc-cnrs.unistra.fr.
authors.forename: Michael
authors.initials: M
authors.lastname: Ryckelynck
mesh_terms: D052157:Aptamers, Nucleotide / Q000138:chemical synthesis / Q000737:chemistry*; D001483:Base Sequence; D001593:Benzyl Compounds / Q000737:chemistry*; D055162:Biocatalysis; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D015723:Gene Library; D057166:High-Throughput Screening Assays; D006801:Humans; D048288:Imidazolines / Q000737:chemistry*; D007700:Kinetics; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D052156:SELEX Aptamer Technique; D014158:Transcription, Genetic
chemical_list: C560120:3,5-difluoro-4-hydroxybenzylidene imidazolinone; D052157:Aptamers, Nucleotide; D001593:Benzyl Compounds; D005456:Fluorescent Dyes; D048288:Imidazolines; D016337:RNA, Catalytic