Direct selection for ribozyme cleavage activity in cells

Xi Chen, Lisa Denison, Matthew Levy, Andrew D. Ellington

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

Selection may prove to be a powerful tool for the generation of functional RNAs for in vivo genetic regulation. However, traditional in vitro selection schemes do not mimic physiological conditions, and in vivo selection schemes frequently use small pool sizes. Here we describe a hybrid in vitro/in vivo selection scheme that overcomes both of these disadvantages. In this new method, PCR-amplified expression templates are transfected into mammalian cells, transcribed hammerhead RNAs self-cleave, and the extracted, functional hammerhead ribozyme species are specifically amplified for the next round of selection. Using this method we have selected a number of cis-cleaving hammerhead ribozyme variants that are functional in vivo and lead to the inhibition of gene expression. More importantly, these results have led us to develop a quantitative, kinetic model that can be used to assess the stringency of the hybrid selection scheme and to direct future experiments. Published by Cold Spring Harbor Laboratory Press.

Original languageEnglish (US)
Pages (from-to)2035-2045
Number of pages11
JournalRNA
Volume15
Issue number11
DOIs
StatePublished - Nov 1 2009

Keywords

  • Hammerhead
  • In vitro selection
  • In vivo kinetics
  • Ribozyme

ASJC Scopus subject areas

  • Molecular Biology

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    Chen, X., Denison, L., Levy, M., & Ellington, A. D. (2009). Direct selection for ribozyme cleavage activity in cells. RNA, 15(11), 2035-2045. https://doi.org/10.1261/rna.1635209