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Nucleic Acids Research Advance Access originally published online on December 1, 2007
Nucleic Acids Research 2008 36(4):e20; doi:10.1093/nar/gkm1062
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Nucleic Acids Research, 2008, Vol. 36, No. 4 e20
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Measuring the dynamic surface accessibility of RNA with the small paramagnetic molecule TEMPOL

Vincenzo Venditti1, Neri Niccolai1 and Samuel E. Butcher2,3,*

1Biomolecular Structure Research Center and Dipartimento di Biologia Molecolare, Università di Siena, via Fiorentina 1, 53100 Siena, Italy, 2Department of Biochemistry and 3Nuclear Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, 433 Babcock Dr. Madison, WI 53706, USA

*To whom correspondence should be addressed. Tel: +1 608 263 3890; Fax: +1 6082623453; Email: butcher{at}biochem.wisc.edu

Received September 11, 2007. Revised November 8, 2007. Accepted November 8, 2007.

The surface accessibility of macromolecules plays a key role in modulating molecular recognition events. RNA is a complex and dynamic molecule involved in many aspects of gene expression. However, there are few experimental methods available to measure the accessible surface of RNA. Here, we investigate the accessible surface of RNA using NMR and the small paramagnetic molecule TEMPOL. We investigated two RNAs with known structures, one that is extremely stable and one that is dynamic. For helical regions, the TEMPOL probing data correlate well with the predicted RNA surface, and the method is able to distinguish subtle variations in atom depths, such as the relative accessibility of pyrimidine versus purine aromatic carbon atoms. Dynamic motions are also detected by TEMPOL probing, and the method accurately reports a previously characterized pH-dependent conformational transition involving formation of a protonated C–A pair and base flipping. Some loop regions are observed to exhibit anomalously high accessibility, reflective of motions that are not evident within the ensemble of NMR structures. We conclude that TEMPOL probing can provide valuable insights into the surface accessibility and dynamics of RNA, and can also be used as an independent means of validating RNA structure and dynamics in solution.


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