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Nucleic Acids Research 2005 33(2):622-631; doi:10.1093/nar/gki182
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Published online 28 January 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org.

Article

Engineered allosteric ribozymes that respond to specific divalent metal ions

Maris Zivarts, Yong Liu and Ronald R. Breaker*

Department of Molecular, Cellular and Developmental Biology, Yale University P.O. Box 208103, New Haven, CT 06520-8103, USA

*To whom correspondence should be addressed. Tel: +1 203 432 9389; Fax: +1 203 432 6604; Email: ronald.breaker{at}yale.edu

Received August 24, 2004. Revised December 1, 2004. Accepted December 20, 2004.

In vitro selection was used to isolate five classes of allosteric hammerhead ribozymes that are triggered by binding to certain divalent metal ion effectors. Each of these ribozyme classes are similarly activated by Mn2+, Fe2+, Co2+, Ni2+, Zn2+ and Cd2+, but their allosteric binding sites reject other divalent metals such as Mg2+, Ca2+ and Sr2+. Through a more comprehensive survey of cations, it was determined that some metal ions (Be2+, Fe3+, Al3+, Ru2+ and Dy2+) are extraordinarily disruptive to the RNA structure and function. Two classes of RNAs examined in greater detail make use of conserved nucleotides within the large internal bulges to form critical structures for allosteric function. One of these classes exhibits a metal-dependent increase in rate constant that indicates a requirement for the binding of two cation effectors. Additional findings suggest that, although complex allosteric functions can be exhibited by small RNAs, larger RNA molecules will probably be required to form binding pockets that are uniquely selective for individual cation effectors.

Present address: Yong Liu, Department of Molecular Biology and Biochemistry, SSB 8166, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada V5A 1S6


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