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. 1995 Dec 11;23(23):4913–4921. doi: 10.1093/nar/23.23.4913

Synthesis and NMR of RNA with selective isotopic enrichment in the bases.

J SantaLucia Jr 1, L X Shen 1, Z Cai 1, H Lewis 1, I Tinoco Jr 1
PMCID: PMC307483  PMID: 8532537

Abstract

Efficient syntheses of pyrimidine and purine nucleosides and nucleotides with selective 13C enrichment in the base moieties are described. Uridine and cytidine are labeled at position C6 and adenosine and guanosine are labeled at position C8. The selectively labeled nucleosides were converted to nucleoside triphosphates and used with in vitro transcription to synthesize labeled RNA. Isotope-edited 12C and 13C sub-spectra of a omega 1-1/2-X-filtered NOESY experiment are demonstrated to be useful for making resonance assignments and for deriving structural information in large (> 20 nt) RNA molecules. The labeled RNAs also allow heteronuclear J-couplings and relaxation parameters to be measured without complications from 13C-13C J-couplings.

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Selected References

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  1. 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]
  2. Chamorro M., Parkin N., Varmus H. E. An RNA pseudoknot and an optimal heptameric shift site are required for highly efficient ribosomal frameshifting on a retroviral messenger RNA. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):713–717. doi: 10.1073/pnas.89.2.713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cowart M., Gibson K. J., Allen D. J., Benkovic S. J. DNA substrate structural requirements for the exonuclease and polymerase activities of procaryotic and phage DNA polymerases. Biochemistry. 1989 Mar 7;28(5):1975–1983. doi: 10.1021/bi00431a004. [DOI] [PubMed] [Google Scholar]
  4. Fesik S. W., Zuiderweg E. R. Heteronuclear three-dimensional NMR spectroscopy of isotopically labelled biological macromolecules. Q Rev Biophys. 1990 May;23(2):97–131. doi: 10.1017/s0033583500005515. [DOI] [PubMed] [Google Scholar]
  5. Marion D., Wüthrich K. Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins. Biochem Biophys Res Commun. 1983 Jun 29;113(3):967–974. doi: 10.1016/0006-291x(83)91093-8. [DOI] [PubMed] [Google Scholar]
  6. Milligan J. F., Groebe D. R., Witherell G. W., Uhlenbeck O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783–8798. doi: 10.1093/nar/15.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Nikonowicz E. P., Pardi A. Three-dimensional heteronuclear NMR studies of RNA. Nature. 1992 Jan 9;355(6356):184–186. doi: 10.1038/355184a0. [DOI] [PubMed] [Google Scholar]
  8. Nikonowicz E. P., Sirr A., Legault P., Jucker F. M., Baer L. M., Pardi A. Preparation of 13C and 15N labelled RNAs for heteronuclear multi-dimensional NMR studies. Nucleic Acids Res. 1992 Sep 11;20(17):4507–4513. doi: 10.1093/nar/20.17.4507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Niu C. H. Synthesis of [4-15NH2]- and [1,3-15N2]cytidine derivatives for use in NMR-monitored binding tests. Anal Biochem. 1984 Jun;139(2):404–407. doi: 10.1016/0003-2697(84)90025-3. [DOI] [PubMed] [Google Scholar]
  10. Ojwang J. O., Hampel A., Looney D. J., Wong-Staal F., Rappaport J. Inhibition of human immunodeficiency virus type 1 expression by a hairpin ribozyme. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10802–10806. doi: 10.1073/pnas.89.22.10802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Otting G., Wüthrich K. Heteronuclear filters in two-dimensional [1H,1H]-NMR spectroscopy: combined use with isotope labelling for studies of macromolecular conformation and intermolecular interactions. Q Rev Biophys. 1990 Feb;23(1):39–96. doi: 10.1017/s0033583500005412. [DOI] [PubMed] [Google Scholar]
  12. Pogolotti A. L., Jr, Ono A., Subramaniam R., Santi D. V. On the mechanism of DNA-adenine methylase. J Biol Chem. 1988 Jun 5;263(16):7461–7464. [PubMed] [Google Scholar]
  13. Puglisi J. D., Wyatt J. R., Tinoco I., Jr Conformation of an RNA pseudoknot. J Mol Biol. 1990 Jul 20;214(2):437–453. doi: 10.1016/0022-2836(90)90192-O. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Serianni A. S., Bondo P. B. 13C-labeled D-ribose: chemi-enzymic synthesis of various isotopomers. J Biomol Struct Dyn. 1994 Apr;11(5):1133–1148. doi: 10.1080/07391102.1994.10508056. [DOI] [PubMed] [Google Scholar]
  15. Shen L. X., Tinoco I., Jr The structure of an RNA pseudoknot that causes efficient frameshifting in mouse mammary tumor virus. J Mol Biol. 1995 Apr 14;247(5):963–978. doi: 10.1006/jmbi.1995.0193. [DOI] [PubMed] [Google Scholar]
  16. Varani G., Tinoco I., Jr RNA structure and NMR spectroscopy. Q Rev Biophys. 1991 Nov;24(4):479–532. doi: 10.1017/s0033583500003875. [DOI] [PubMed] [Google Scholar]
  17. Webb T. R., Matteucci M. D. Hybridization triggered cross-linking of deoxyoligonucleotides. Nucleic Acids Res. 1986 Oct 10;14(19):7661–7674. doi: 10.1093/nar/14.19.7661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Williamson J. R., Boxer S. G. Multinuclear NMR studies of DNA hairpins. 1. Structure and dynamics of d(CGCGTTGTTCGCG). Biochemistry. 1989 Apr 4;28(7):2819–2831. doi: 10.1021/bi00433a012. [DOI] [PubMed] [Google Scholar]
  19. Yoshikawa M., Kato T., Takenishi T. A novel method for phosphorylation of nucleosides to 5'-nucleotides. Tetrahedron Lett. 1967 Dec;50:5065–5068. doi: 10.1016/s0040-4039(01)89915-9. [DOI] [PubMed] [Google Scholar]

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