HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 282, Issue 49, 35510-35518, December 7, 2007

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 282/49/35510    most recent M706993200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Donini, S. Articles by Peracchi, A. Search for Related Content PubMed PubMed Citation Articles by Donini, S. Articles by Peracchi, A. Social Bookmarking                      What's this?

The Advantages of Being Locked

ASSESSING THE CLEAVAGE OF SHORT AND LONG RNAs BY LOCKED NUCLEIC ACID-CONTAINING 8–17 DEOXYRIBOZYMES^*

Stefano Donini¹, Marcello Clerici, Jesper Wengel, Birte Vester^¶2, and Alessio Peracchi³

From the Department of Biochemistry and Molecular Biology, University of Parma, 43100 Parma, Italy, ^¶Nucleic Acid Center, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark, and Nucleic Acid Center, Department of Chemistry, University of Southern Denmark, DK-5230 Odense M, Denmark

RNA-cleaving deoxyribozymes can be used for the sequence-specific knockdown of mRNAs. It was previously shown that activity of these deoxyribozymes is enhanced when their substrate-binding arms include some locked nucleic acid (LNA) residues, but the mechanistic basis of this enhancement was not explored. Here we dissected the kinetics and thermodynamics underlying the reaction of LNA-containing 8–17 deoxyribozymes. Four 8–17 constructs were designed to target sequences within the E6 mRNA from human papillomavirus type 16. When one of these deoxyribozymes (DNAzymes) and the corresponding LNA-armed enzyme (LNAzyme) were tested against a minimal RNA substrate, they showed similar rates of substrate binding and similar rates of intramolecular cleavage, but the LNAzyme released its substrate more slowly. The superior thermodynamic stability of the LNAzyme-substrate complex led to improved performances in reactions carried out at low catalyst concentrations. The four DNAzymes and the corresponding LNAzymes were then tested against extended E6 transcripts (>500 nucleotides long). With these structured substrates, the LNAzymes retained full activity, whereas the DNAzymes cleaved extremely poorly, unless they were allowed to pre-anneal to their targets. These results imply that LNAzymes can easily overcome the kinetic barrier represented by local RNA structure and bind to folded targets with a faster association rate as compared with DNAzymes. Such faster annealing to structured targets can be explained by a model whereby LNA monomers favor the initial hybridization to short stretches of unpaired residues ("nucleation"), which precedes disruption of the local mRNA structure and completion of the binding process.

Received for publication, August 21, 2007 , and in revised form, September 24, 2007.

^* This work was supported in part by grants from the Danish National Research Foundation (to J. W. and B. V.) and the EMBO Young Investigator Programme (to A. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–4 and Table S1.

¹ Supported by a Marie Curie Early Stage Research Training Fellowship of the European Community's Sixth Framework Programme under Contract Number MEST-CT-2004-504018.

² To whom correspondence may be addressed. Tel.: 45-6550-2377; Fax: 45-6550-2467; E-mail: b.vester{at}bmb.sdu.dk. ³ To whom correspondence may be addressed. Tel.: 39-0521-905137; Fax: 39-0521-905151; E-mail: peracchi{at}unipr.it.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				K. R. Julien, M. Sumita, P.-H. Chen, I. A. Laird-Offringa, and C. G. Hoogstraten Conformationally restricted nucleotides as a probe of structure-function relationships in RNA RNA, August 1, 2008; 14(8): 1632 - 1643. [Abstract] [Full Text] [PDF]