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Journal of Biochemistry Advance Access originally published online on January 4, 2009
Journal of Biochemistry 2009 145(4):451-459; doi:10.1093/jb/mvn182
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Highly Active Low Magnesium Hammerhead Ribozyme

Agnieszka Fedoruk-Wyszomirska*, Maciej Szymanski*, Eliza Wyszko, Miroslawa Z. Barciszewska and Jan Barciszewski{dagger}

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland

{dagger}To whom correspondence should be addressed. Tel: +48-61-8528503, Fax: +48-61-8530523, E-mail: jan.barciszewski{at}ibch.poznan.pl

Received October 28, 2008; Accepted December 19, 2008


  Abstract

Hammerhead (HH) ribozymes can be used for highly specific inhibition of gene expression through the degradation of target mRNA. In vitro experiments with minimal HH domains demonstrated that the efficiency of catalysis is highly dependent on concentration of magnesium ions. Optimal ion requirements for HH-catalysed RNA cleavage are far from these found in the cell. Recently, it has been proposed that the efficiency of HH ribozymes can be increased at low magnesium concentration through stabilization of a catalytically active conformation by tertiary interactions between helices I and II. We designed a ribozyme stabilized by GAAA tetraloop and its receptor motifs and demonstrated that it can efficiently catalyse target RNA hydrolysis at submillimolar Mg2+ concentrations in vitro as well as in cultured cells. Both unmodified and locked nucleic acid-modified extended ribozymes proved superior to the minimal core ribozyme and DNAzyme against the same target sequence.

Key Words: DNAzyme, hammerhead ribozyme, HIV, RNA, tetraloop receptor

Abbreviations: HH, hammerhead; TL, tetraloop; TLR, tetraloop receptor

*These authors contributed equally to this work.


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