Journal of Biomolecular NMR

, Volume 35, Issue 4, pp 261–274 | Cite as

Alternate-site isotopic labeling of ribonucleotides for NMR studies of ribose conformational dynamics in RNA

  • James E. JohnsonJr
  • Kristine R. Julien
  • Charles G. Hoogstraten


Heteronuclear NMR spin relaxation studies of conformational dynamics are coming into increasing use to help understand the functions of ribozymes and other RNAs. Due to strong \(^{13}\hbox{C}--^{13}\hbox{C}\) magnetic interactions within the ribose ring, however, these studies have thus far largely been limited to 13C and 15N resonances on the nucleotide base side chains. We report here the application of the alternate-site 13C isotopic labeling scheme, pioneered by LeMaster for relaxation studies of amino acid side chains, to nucleic acid systems. We have used different strains of E. coli to prepare mononucleotides containing 13C label in one of two patterns: Either C1′ or C2′ in addition to C4′, termed (1′/2′,4′) labeling, or nearly complete labeling at the C2′ and C4′ sites only, termed (2′,4′) labeling. These patterns provide isolated \(^{13}\hbox{C}--^{1}\)H spin systems on the labeled carbon atoms and thus allow spin relaxation studies without interference from \(^{13}\hbox{C}--^{13}\hbox{C}\) scalar or dipolar coupling. Using relaxation studies of AMP dissolved in glycerol at varying temperature to produce systems with correlation times characteristic of different size RNAs, we demonstrate the removal of errors due to \(^{13}\hbox{C}--^{13}\hbox{C}\) interaction in T 1 measurements of larger nucleic acids and in T measurements in RNA molecules. By extending the applicability of spin relaxation measurements to backbone ribose groups, this technology should greatly improve the flexibility and completeness of NMR analyses of conformational dynamics in RNA.

Key words

alternate-site dynamics glucose-6-phosphate dehydrogenase isotope labeling ribose RNA 



adenosine 5′-monophosphate


cytidine 5′-monophosphate




chemical shift anisotropy






glucose-6-phosphate dehydrogenase


heteronuclear single-quantum correlation




ribonucleoside 5′-monophosphate


tricarboxylic acid


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The authors are grateful to David LeMaster for helpful discussions, to John SantaLucia for comments on the manuscript, and to the Coli Genetic Stock Center (Yale) for bacterial strains. This work was supported by faculty startup funds and an Intramural Research Program Grant from Michigan State University and by the National Institutes of Health (GM-069742).


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Copyright information

© Springer 2006

Authors and Affiliations

  • James E. JohnsonJr
    • 1
  • Kristine R. Julien
    • 1
  • Charles G. Hoogstraten
    • 1
  1. 1.Department of Biochemistry & Molecular BiologyMichigan State UniversityEast LansingUSA

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