Journal
RNA
Volume 15, Issue 11, Pages 2035-2045Publisher
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.1635209
Keywords
hammerhead; ribozyme; in vitro selection; in vivo kinetics
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Funding
- National Institutes of Health [R01 GM077040]
- Welch Foundation [F-1654]
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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.
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