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Preparation of Specifically Deuterated and 13C-Labeled RNA for NMR Studies Using Enzymatic Synthesis

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    Preparation of Specifically Deuterated and 13C-Labeled RNA for NMR Studies Using Enzymatic Synthesis†
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    Contribution from the Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
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    Journal of the American Chemical Society

    Cite this: J. Am. Chem. Soc. 1997, 119, 50, 12100–12108
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    https://doi.org/10.1021/ja9725054
    Published December 17, 1997
    Copyright © 1997 American Chemical Society
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    Abstract

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    The enzymatic conversion of glucose into ATP, GTP, UTP, and CTP with several different isotopic labeling patterns is described. Enzymes of the pentose phosphate pathway and enzyme-catalyzed hydrogen exchange were used to convert three types of isotopically labeled glucose into [1‘,2‘,3‘,4‘,5‘,5‘-2H6]NTPs (1−4), [3‘,4‘,5‘,5‘-2H4]UTP (5), [1‘,2‘,3‘,4‘,5‘-13C5]NTPs (6−9), and [3‘,4‘,5‘,5‘-2H4-1‘,2‘,3‘,4‘,5‘-13C5]NTPs (10−13), which were then used to synthesize a 30 nucleotide HIV TAR RNA. Representative NOESY and HSQC spectra were acquired to demonstrate the utility of the new labeling patterns. The spectral editing afforded by 2H and 13C labeling dramatically simplifies the crowded NOESY and HSQC spectra of RNA molecules. The synthetic methods described here will permit the preparation of several specifically deuterated and/or 13C-labeled forms of RNA which should be useful in NMR structural studies of large RNAs.

    Copyright © 1997 American Chemical Society

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    †

     Abbreviations used:  NTP, nucleoside triphosphate; NOE, nuclear Overhauser effect; PRPP, 5-phospho-d-ribosyl α-1-pyrophosphate; PEP, phosphoenolpyruvate; s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; calcd, calculated; PCR, polymerase chain reaction; IPTG, isopropyl β-d-thiogalactopyranoside; LB medium, Luria−Bertani medium; OD, optical density; UPRT, uracil phosphoribosyltransferase; APRT, adenine phosphoribosyltransferase; XGPRT, xanthine−guanine phosphoribosyltransferase; TEABC, triethylammonium bicarbonate; 3PGA, 3-phosphoglycerate; PPi, inorganic pyrophosphate; DTT, dithiothreitol; Glu, glucose; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; 6PG, 6-phosphogluconate; Ru5P, ribulose-5-phosphate; R5P, ribose-5-phosphate; d6-NTPs, [1‘,2‘,3‘,4‘,5‘,5‘-2H4]NTPs; d4-NTPs, [3‘,4‘,5‘,5‘-2H4]NTPs; 13C5-ribose-NTPs, [1‘,2‘,3‘,4‘,5‘-13C5]NTPs; d4-13C5-ribose-NTPs, [3‘,4‘,5‘,5‘-2H4-1‘,2‘,3‘,4‘,5‘-13C5]NTPs; d6-RNA, RNA prepared from d6-NTPs (1−4); d4/d6-RNA, RNA prepared from d4-UTP (5) and d6-NTPs (1, 2, 4); 13C5-ribose-RNA, RNA prepared from 13C5-ribose-NTPs (6−9); d4-13C5-ribose-RNA, RNA prepared from d4-13C5-ribose-NTPs (10−13).

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    In papers with more than one author, the asterisk indicates the name of the author to whom inquiries about the paper should be addressed.

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     Abstract published in Advance ACS Abstracts, November 15, 1997.

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    Cite this: J. Am. Chem. Soc. 1997, 119, 50, 12100–12108
    Click to copy citationCitation copied!
    https://doi.org/10.1021/ja9725054
    Published December 17, 1997
    Copyright © 1997 American Chemical Society
    Request reuse permissions

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