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Ribozymes pp 13-24 | Cite as

Co-transcriptional Analysis of Self-Cleaving Ribozymes and Their Ligand Dependence

  • Luiz F. M. Passalacqua
  • Andrej LuptákEmail author
Protocol
  • 66 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2167)

Abstract

Self-cleaving ribozymes are RNA molecules that catalyze a site-specific self-scission reaction. Analysis of self-cleavage is a crucial aspect of the biochemical study and understanding of these molecules. Here we describe a co-transcriptional assay that allows the analysis of self-cleaving ribozymes in different reaction conditions and in the presence of desired ligands and/or cofactors. Utilizing a standard T7 RNA polymerase in vitro transcription system under limiting Mg2+ concentration, followed by a 25-fold dilution of the reaction in desired conditions of self-cleavage (buffer, ions, ligands, pH, temperature, etc.) to halt the synthesis of new RNA molecules, allows the study of self-scission of these molecules without the need for purification or additional preparation steps, such as refolding procedures. Furthermore, because the transcripts are not denatured, this assay likely yields RNAs in conformations relevant to co-transcriptionally folded species in vivo.

Key words

Ribozymes Catalytic RNA Self-cleaving ribozymes Co-transcriptional analysis Co-transcriptional kinetics In vitro transcription Metal-ion dependence Ligand dependence 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  1. 1.Department of Pharmaceutical SciencesUniversity of CaliforniaIrvineUSA
  2. 2.Department of Molecular Biology and BiochemistryUniversity of CaliforniaIrvineUSA
  3. 3.Department of ChemistryUniversity of CaliforniaIrvineUSA

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