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

Abstract

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.

Partial 13C isotopic enrichment of nucleoside monophosphates: useful reporters for NMR structural studies

“…Protocols to extract intact RNA from cells grown on uniformly isotopically labeled media and to isolate nucleoside monophosphates (NMPs) from the RNA have been described (6,14), but the use of specifically labeled precursors is less common (7,15). Isolating deoxyNMPs (dNMPs) is more difficult since cells contain 7–10 times more RNA than DNA (16), but this, too, has been described (17,18).…”

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

“…For example deuterium labels are introduced at carbons 3ʹ′, 4ʹ′, 5ʹ′ resulting in non-exchangeable protons at H1ʹ′, H2ʹ′ and at the base positions H6, H8, H2 [203]. This can also be combined with 13 C labeling of the entire ribose [204].…”

The Synthesis of Deuterionucleosides

“…In a recent paper 141 isomerase. The reactions are conducted in a one-pot fashion followed by boronate chromatography.…”

“…For examples, labelling the prochiral 5'-methylene moiety for stereospecific assignment and through this to measure vicinal 1 H-31 P coupling constants 163 ; to improve the accuracy of coupling data extracted from 1 H-13 C HSQC spectra 222 ; to eliminate crosspeaks completely from crowded regions of 2D spectra 141 or to aid relaxation time measurements 43,46,223,228,229 can be recalled.…”