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Nucleic Acids Research 2005 33(18):5763-5770; doi:10.1093/nar/gki877
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Published online 12 October 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}oxfordjournals.org

Article

Molecular beacons as probes of RNA unfolding under native conditions

Julia F. Hopkins and Sarah A. Woodson1,*

Cell, Molecular and Developmental Biology and Biophysics Program, Johns Hopkins University 3400 N. Charles St., Baltimore, MD 21218-2685, USA 1T.C. Jenkins Department of Biophysics, Johns Hopkins University 3400 N. Charles St., Baltimore, MD 21218-2685, USA

*To whom correspondence should be addressed. Tel: +1 410 516 2015; Fax: +1 410 516 4118; Email: swoodson{at}jhu.edu

Received August 18, 2005. Revised September 14, 2005. Accepted September 14, 2005.

Hybridization of fluorescent molecular beacons provides real-time detection of RNA secondary structure with high specificity. We used molecular beacons to measure folding and unfolding rates of the Tetrahymena group I ribozyme under native conditions. A molecular beacon targeted against 15 nt in the 5' strand of the P3 helix specifically hybridized with misfolded forms of the ribozyme, without invading the native tertiary structure. The beacon associated with the misfolded ribozyme 300 times more slowly than with an unstructured oligonucleotide containing the same target sequence, suggesting that the misfolded ribozyme core remains structured in the absence of Mg2+. The rate of beacon hybridization under native conditions revealed a linear relationship between the free energy of unfolding and Mg2+ concentration. A small fraction of the RNA population unfolded very rapidly, suggesting parallel unfolding in one step or through misfolded intermediates.

Correspondence may also be addressed to Julia F. Hopkins. Tel: +1 410 516 6776; Fax: +1 410 516 4118; Email: jfhopkins{at}jhu.edu


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