Kramat, Janice ; Suess, Beatrix (2022)
Efficient Method to Identify Synthetic Riboswitches Using RNA-Based Capture-SELEX Combined with In Vivo Screening.
In: Methods in molecular biology (Clifton, N.J.), 2518
doi: 10.1007/978-1-0716-2421-0_10
Artikel, Bibliographie
Kurzbeschreibung (Abstract)
Synthetic riboswitches are a promising tool for conditional gene expression. In vitro selected aptamers used as binding domains for the design of RNA-based switches have to exhibit excellent binding affinity as well as ligand binding-induced structural changes. Selection via Capture-SELEX favors the enrichment of aptamers which exhibit both characteristics. For the Capture-SELEX, an RNA pool is used that gets immobilized onto a capture oligonucleotide by hybridization. Addition of the ligand frees the aptamers by their binding to the ligand, resulting in the release from the capture oligonucleotide through structural changes. These sequences get reverse transcribed, PCR amplified, and used for the following selection rounds. In this publication, we present a detailed protocol for Capture-SELEX, followed by screening in yeast to identify aptamers suitable for the design of synthetic riboswitches.
| Typ des Eintrags: | Artikel |
|---|---|
| Erschienen: | 2022 |
| Autor(en): | Kramat, Janice ; Suess, Beatrix |
| Art des Eintrags: | Bibliographie |
| Titel: | Efficient Method to Identify Synthetic Riboswitches Using RNA-Based Capture-SELEX Combined with In Vivo Screening |
| Sprache: | Englisch |
| Publikationsjahr: | 2022 |
| Titel der Zeitschrift, Zeitung oder Schriftenreihe: | Methods in molecular biology (Clifton, N.J.) |
| Jahrgang/Volume einer Zeitschrift: | 2518 |
| DOI: | 10.1007/978-1-0716-2421-0_10 |
| Kurzbeschreibung (Abstract): | Synthetic riboswitches are a promising tool for conditional gene expression. In vitro selected aptamers used as binding domains for the design of RNA-based switches have to exhibit excellent binding affinity as well as ligand binding-induced structural changes. Selection via Capture-SELEX favors the enrichment of aptamers which exhibit both characteristics. For the Capture-SELEX, an RNA pool is used that gets immobilized onto a capture oligonucleotide by hybridization. Addition of the ligand frees the aptamers by their binding to the ligand, resulting in the release from the capture oligonucleotide through structural changes. These sequences get reverse transcribed, PCR amplified, and used for the following selection rounds. In this publication, we present a detailed protocol for Capture-SELEX, followed by screening in yeast to identify aptamers suitable for the design of synthetic riboswitches. |
| ID-Nummer: | pmid:35666445 |
| Fachbereich(e)/-gebiet(e): | 10 Fachbereich Biologie 10 Fachbereich Biologie > Synthetic RNA biology |
| Hinterlegungsdatum: | 14 Jun 2022 06:26 |
| Letzte Änderung: | 25 Jul 2023 08:25 |
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