Preparation of partially 2H/13C-labelled RNA for NMR studies. Stereo-specific deuteration of the H5" in nucleotides.
An effective in vitro enzymatic synthesis is described for the production of nucleoside triphosphates (NTPs) which are stereo-specifically deuterated on the H5" position with high selectivity (>98%), and which can have a variety of different labels (13C, 15N, 2H) in other positions. The NTPs can subsequently be employed in the enzymatic synthesis of RNAs using T7 polymerase from a DNA template. The stereo-specific deuteration of the H5" immediately provides the stereo-specific assignment of H5' resonances in NMR spectra, giving access to important structural parameters. Stereo-chemical H-exchange was used to convert commercially available 1,2,3,4,5,6,6-2H-1,2,3,4,5,6-13C-D-glucose (d7-13C6-D-glucose) into [1,2,3,4,5,6(R)-2H-1,2,3,4,5,6-13C]-D-glucose (d6-13C6-D-glucose). [1',3',4',5"-2H-1',2',3',4',5'-13C]GTP (d4-13C5-GTP) was then produced from d6-13C6-D-glucose and guanine base via in vitro enzymatic synthesis employing enzymes from the pentose-phosphate, nucleotide biosynthesis and salvage pathways. The overall yield was approximately 60 mg NTP per 1 g glucose, comparable with the yield of NTPs isolated from Escherichia coli grown on enriched media. The d4-13C5-GTP, together with in vitro synthesised d5-UTP, d5-CTP and non-labelled ATP, were used in the synthesis of a 31 nt RNA derived from the primer binding site of hepatitis B virus genomic RNA. (13C,1H) hetero-nuclear multiple-quantum spectra of the specifically deuterated sample and of a non-deuterated uniformly 13C/15N-labelled sample demonstrates the reduced spectral crowding and line width narrowing compared with 13C-labelled non-deuterated RNA.
Cromsigt
Jenny
J
Department of Medical Biochemistry and Biophysics and Organic Chemistry, Umeå University, S 901 87 Umeå, Sweden.
Schleucher
Jürgen
J
Gustafsson
Tomas
T
Kihlberg
Jan
J
Wijmenga
Sybren
S
D002247:Carbon Isotopes / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry; D006515:Hepatitis B virus / Q000235:genetics; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009843:Oligoribonucleotides / Q000737:chemistry / Q000235:genetics; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D012265:Ribonucleotides / Q000737:chemistry / Q000235:genetics
D002247:Carbon Isotopes; D009843:Oligoribonucleotides; D012265:Ribonucleotides; D012313:RNA; D003903:Deuterium