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Nucleic Acids Research Advance Access published online on June 26, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn397
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© 2008 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

Multiple segmental and selective isotope labeling of large RNA for NMR structural studies

Frank H. T. Nelissen, Adriaan J. van Gammeren, Marco Tessari, Frederic C. Girard, Hans A. Heus and Sybren S. Wijmenga*

Department of Biophysical Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands

*To whom correspondence should be addressed. Tel: +31 24 3652678; Fax: +31 24 3652112; Email: s.wijmenga{at}nmr.ru.nl

Received April 15, 2008. Revised June 5, 2008. Accepted June 5, 2008.

Multiple segmental and selective isotope labeling of RNA with three segments has been demonstrated by introducing an RNA segment, selectively labeled with 13C9/15N2/2H(1', 3', 4', 5', 5'')-labeled uridine residues, into the central position of the 20 kDa {epsilon}-RNA of Duck Hepatitis B Virus. The RNA molecules were produced via two efficient protocols: a two-step protocol, which uses T4 DNA ligase and T4 RNA ligase 1, and a one-pot protocol, which uses T4 RNA ligase 1 alone. With T4 RNA ligase 1 all not-to-be-ligated termini are usually protected to prevent formation of side products. We show that such labor-intensive protection of termini is not required, provided segmentation sites can be chosen such that the segments fold into the target structure or target-like structures and thus are not trapped into stable alternate structures. These sites can be reliably predicted via DINAMelt. The simplified NMR spectrum provided evidence for the presence of a U28 H3-imino resonance, previously obscured in the fully labeled sample, and thus of the non-canonical base pair U28:C37. The demonstrated multiple segmental labeling protocols are generally applicable to large RNA molecules and can be extended to more than three segments.


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F. H. T. Nelissen, F. C. Girard, M. Tessari, H. A. Heus, and S. S. Wijmenga
Preparation of selective and segmentally labeled single-stranded DNA for NMR by self-primed PCR and asymmetrical endonuclease double digestion
Nucleic Acids Res., September 1, 2009; 37(17): e114 - e114.
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