Preparation of specifically deuterated and 13C-labeled RNA for NMR studies using enzymatic synthesis

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'-H-2(6)]NTPs (1-4), [3',4',5',5'-H-2(4)]UTP (5), [1',2',3',4',5'-C-13(5)]NTPs (6-9), and [3',4',5',5'-H-2(4)-1',2',3',4',5'-C-13(5)]NTP (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 H-2 and C-13 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 C-13-labeled forms of RNA which should be useful in NMR structural studies of large RNAs.