Engineering high-speed allosteric hammerhead ribozymes

Kristian H. Link; Lixia Guo; Tyler D. Ames; Laising Yen; Richard C. Mulligan; Ronald R. Breaker

doi:10.1515/bc.2007.105

journal article Aug 01, 2007

Engineering high-speed allosteric hammerhead ribozymes

Kristian H. Link Lixia Guo

Tyler D. Ames Laising Yen Richard C. Mulligan Ronald R. Breaker

bchm Vol. 388 No. 8 pp. 779-786 · Walter de Gruyter GmbH

View at Publisher Save 10.1515/bc.2007.105

Abstract

Abstract
Full-length hammerhead ribozymes were subjected to in vitro selection to identify variants that are allosterically regulated by theophylline in the presence of a physiologically relevant concentration of Mg2+. The population of allosteric ribozymes resulting from 15 rounds of in vitro selection yielded variants with observed rate constants (k
obs) as high as 8 min-1 in the presence of theophylline and maximal k
obs increases of up to 285-fold compared to rate constants measured in the absence of effector. The selected ribozymes have kinetic characteristics that are predicted to be sufficient for cellular gene control applications, but do not exhibit any activity in reporter gene assays. The inability of the engineered RNAs to control gene expression suggests that the in vitro and in vivo folding pathways of the RNAs are different. These results provide several key pieces of information that will aid in future efforts to engineer allosteric ribozymes for gene control applications.

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Citations

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References

Details

Published: Aug 01, 2007
Vol/Issue: 388(8)
Pages: 779-786

Authors

K

Kristian H. Link

Howard Hughes Medical Institute, Yale University, New Haven, CT 06520-8103, USA

L

Lixia Guo

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA

T

Tyler D. Ames

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA

L

Laising Yen

Department of Genetics, Harvard Institute of Human Genetics, Harvard Medical School, Boston, MA 02115, USA

R

Richard C. Mulligan

Department of Genetics, Harvard Institute of Human Genetics, Harvard Medical School, Boston, MA 02115, USA

R

Ronald R. Breaker

Howard Hughes Medical Institute, Yale University, New Haven, CT 06520-8103, USA, Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA and Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8103, USA

Cite This Article

Kristian H. Link, Lixia Guo, Tyler D. Ames, et al. (2007). Engineering high-speed allosteric hammerhead ribozymes. bchm, 388(8), 779-786. https://doi.org/10.1515/bc.2007.105

Engineering high-speed allosteric hammerhead ribozymes

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