Abstract
Isotopic labeling of RNA with 13C and 15N has become a routine procedure in structural studies by NMR spectroscopy. The methodology in this paper describes the random fractional deuteration of RNA using the obligate methylotropic bacterium, Methylophilus methylotrophus. This bacterium was grown using a non-deuterated carbon source in 52:48 D20/H20 and we have shown that all protons in the ribonucleotides except for the ribose H1 become 52% randomly fractionally deuterated. Improved growth conditions for this organism are also described that yield higher cell densities in liquid culture, which is applicable for all labeling procedures.
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- Batey R. T., Battiste J. L., Williamson J. R. Preparation of isotopically enriched RNAs for heteronuclear NMR. Methods Enzymol. 1995;261:300–322. doi: 10.1016/s0076-6879(95)61015-4. [DOI] [PubMed] [Google Scholar]
- Batey R. T., Inada M., Kujawinski E., Puglisi J. D., Williamson J. R. Preparation of isotopically labeled ribonucleotides for multidimensional NMR spectroscopy of RNA. Nucleic Acids Res. 1992 Sep 11;20(17):4515–4523. doi: 10.1093/nar/20.17.4515. [DOI] [PMC free article] [PubMed] [Google Scholar]
- CHORNEY W., SCULLY N. J., CRESPI H. L., KATZ J. J. The growth of algae in deuterium oxide. Biochim Biophys Acta. 1960 Jan 15;37:280–287. doi: 10.1016/0006-3002(60)90235-3. [DOI] [PubMed] [Google Scholar]
- LeMaster D. M. Deuteration in protein proton magnetic resonance. Methods Enzymol. 1989;177:23–43. doi: 10.1016/0076-6879(89)77004-x. [DOI] [PubMed] [Google Scholar]
- LeMaster D. M., Richards F. M. NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteriation. Biochemistry. 1988 Jan 12;27(1):142–150. doi: 10.1021/bi00401a022. [DOI] [PubMed] [Google Scholar]
- Moore P. B. The preparation of deuterated ribosomal materials for neutron scattering. Methods Enzymol. 1979;59:639–655. doi: 10.1016/0076-6879(79)59119-8. [DOI] [PubMed] [Google Scholar]