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Direct in vitro selection of trans-acting ribozymes for posttranscriptional, site-specific, and covalent fluorescent labeling of RNA

Please always quote using this URN: urn:nbn:de:bvb:20-opus-192333
  • General and efficient tools for site-specific fluorescent or bioorthogonal labeling of RNA are in high demand. Here, we report direct in vitro selection, characterization, and application of versatile trans-acting 2'-5' adenylyl transferase ribozymes for covalent and site-specific RNA labeling. The design of our partially structured RNA pool allowed for in vitro evolution of ribozymes that modify a predetermined nucleotide in cis (i.e. intramolecular reaction), and were then easily engineered for applications in trans (i.e. in an intermolecularGeneral and efficient tools for site-specific fluorescent or bioorthogonal labeling of RNA are in high demand. Here, we report direct in vitro selection, characterization, and application of versatile trans-acting 2'-5' adenylyl transferase ribozymes for covalent and site-specific RNA labeling. The design of our partially structured RNA pool allowed for in vitro evolution of ribozymes that modify a predetermined nucleotide in cis (i.e. intramolecular reaction), and were then easily engineered for applications in trans (i.e. in an intermolecular setup). The resulting ribozymes are readily designed for specific target sites in small and large RNAs and accept a wide variety of N6-modified ATP analogues as small molecule substrates. The most efficient new ribozyme (FH14) shows excellent specificity towards its target sequence also in the context of total cellular RNA.show moreshow less
Metadaten
Author: Mohammad Ghaem Maghami, Carolin P. M. Scheitl, Claudia HöbartnerORCiD
URN:urn:nbn:de:bvb:20-opus-192333
Document Type:Preprint
Faculties:Fakultät für Chemie und Pharmazie / Institut für Organische Chemie
Language:English
Parent Title (English):Journal of the American Chemical Society
Year of Completion:2019
Source:Journal of the American Chemical Society 2019, https://doi.org/10.1021/jacs.9b10531 Copyright © 2019 American Chemical Society
DOI:https://doi.org/10.1021/jacs.9b10531
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 54 Chemie / 547 Organische Chemie
Tag:Ribozyme-catalyzed RNA labeling; covalent and site-specific RNA labeling; in vitro selection from a structured RNA library; intermolecular applications of ribozymes; trans-acting 2'-5' adenylyl transferase ribozymes
Release Date:2019/12/02
Note:
This document is the unedited Author’s version of a Submitted Work that was subsequently accepted for publication in Journal of the American Chemical Society, copyright © American Chemical Society after peer review. To access the final edited and published work see https://doi.org/10.1021/jacs.9b10531.
EU-Project number / Contract (GA) number:682586
OpenAIRE:OpenAIRE
Licence (German):License LogoDeutsches Urheberrecht