Preparation of specifically deuterated RNA for NMR studies using a combination of chemical and enzymatic synthesis

The syntheses of ATP, GTP, UTP, and CTP with deuterium labels on the 3', 4', and 5' carbons (2-5) is described. A combination of chemical and enzymatic synthesis is used where D,L-ribose-3,4,5,5'-d(4) (+/-1) is first produced from glycerol-d(8) by chemical methods, and then the four 3',4',5',5'-labeled NTPs (2-5) are prepared from (-1) using enzymes from the purine salvage and pyrimidine biosynthetic metabolic pathways. New procedures were developed for the large scale preparation of GTP and CTP, and existing procedures were modified for the preparation of ATP and UTP. A 30-nucleotide RNA derived from the HIV-2 TAR RNA was prepared with unlabeled NTPs and deuterated NTPs (2-5) to illustrate the dramatic effects of deuteration on the NMR spectra of RNA. The NOESY spectra of the deuterated RNA exhibits greatly reduced spectral crowding compared to that of the unlabeled RNA, and assignment of NOEs to the H2' protons is simplified due to the specific deuteration pattern. Also, the nonselective T-1 and T-2 relaxation rates were measured for the deuterated RNA and found to be approximately twice as long as the T-1 and T-2 relaxation rates of the unlabeled RNA. The spectral simplification and improved relaxation properties of the deuterated RNA should prove useful in the study of large RNAs by NMR.