title: Using droplet-based microfluidics to improve the catalytic properties of RNA under multiple-turnover conditions.
abstract: In vitro evolution methodologies are powerful approaches to identify RNA with new functionalities. While Systematic Evolution of Ligands by Exponential enrichment (SELEX) is an efficient approach to generate new RNA aptamers, it is less suited for the isolation of efficient ribozymes as it does not select directly for the catalysis. In vitro compartmentalization (IVC) in aqueous droplets in emulsions allows catalytic RNAs to be selected under multiple-turnover conditions but suffers severe limitations that can be overcome using the droplet-based microfluidics workflow described in this paper. Using microfluidics, millions of genes in a library can be individually compartmentalized in highly monodisperse aqueous droplets and serial operations performed on them. This allows the different steps of the evolution process (gene amplification, transcription, and phenotypic assay) to be uncoupled, making the method highly flexible, applicable to the selection and evolution of a variety of RNAs, and easily adaptable for evolution of DNA or proteins. To demonstrate the method, we performed cycles of random mutagenesis and selection to evolve the X-motif, a ribozyme which, like many ribozymes selected using SELEX, has limited multiple-turnover activity. This led to the selection of variants, likely to be the optimal ribozymes that can be generated using point mutagenesis alone, with a turnover number under multiple-turnover conditions, k(ss) cat, ∼ 28-fold higher than the original X-motif, primarily due to an increase in the rate of product release, the rate-limiting step in the multiple-turnover reaction.
authors.affiliation: Architecture et Réactivité de l'ARN, CNRS UPR 9002, Université de Strasbourg, 67084 Strasbourg, France Institut de Science et d'Ingénierie Supramoléculaires (ISIS), CNRS UMR 7006, Université de Strasbourg, 67083 Strasbourg, France andrew.griffiths@espci.fr m.ryckelynck@unistra.fr.
authors.forename: Michael
authors.initials: M
authors.lastname: Ryckelynck
authors.affiliation: Architecture et Réactivité de l'ARN, CNRS UPR 9002, Université de Strasbourg, 67084 Strasbourg, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Baudrey
authors.affiliation: Architecture et Réactivité de l'ARN, CNRS UPR 9002, Université de Strasbourg, 67084 Strasbourg, France Institut de Science et d'Ingénierie Supramoléculaires (ISIS), CNRS UMR 7006, Université de Strasbourg, 67083 Strasbourg, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Rick
authors.affiliation: Institut de Science et d'Ingénierie Supramoléculaires (ISIS), CNRS UMR 7006, Université de Strasbourg, 67083 Strasbourg, France.
authors.forename: Annick
authors.initials: A
authors.lastname: Marin
authors.affiliation: Institut de Science et d'Ingénierie Supramoléculaires (ISIS), CNRS UMR 7006, Université de Strasbourg, 67083 Strasbourg, France.
authors.forename: Faith
authors.initials: F
authors.lastname: Coldren
authors.affiliation: Architecture et Réactivité de l'ARN, CNRS UPR 9002, Université de Strasbourg, 67084 Strasbourg, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Westhof
authors.affiliation: Institut de Science et d'Ingénierie Supramoléculaires (ISIS), CNRS UMR 7006, Université de Strasbourg, 67083 Strasbourg, France Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI ParisTech), CNRS UMR 8231, 75231 Paris, France andrew.griffiths@espci.fr m.ryckelynck@unistra.fr.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Griffiths
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics*; D004247:DNA / Q000235:genetics; D019020:Directed Molecular Evolution; D044085:Microfluidics; D016337:RNA, Catalytic / Q000235:genetics* / Q000302:isolation & purification; D052156:SELEX Aptamer Technique
chemical_list: D052157:Aptamers, Nucleotide; D016337:RNA, Catalytic; D004247:DNA
keywords: RNA; droplet-based microfluidics; high-throughput screening; in vitro evolution; ribozymes