Preparation of Specifically Deuterated RNA for NMR Studies Using a Combination of Chemical and Enzymatic Synthesis

Abstract

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.

A Nano-Chip-LC/MSn Based Strategy for Characterization of Modified Nucleosides Using Reduced Porous Graphitic Carbon as a Stationary Phase

“…The test-mixture contained the following 13 nucleosides in varying amounts (Table 1, [25], inorganic pyrophosphatase (hppA, EC 3.6.1.1) [26], and CTP synthetase (pyrG, EC 6.3.4.2) [27], were prepared from overexpression strains described. All standard analytical HPLC methods and stepwise purification methods are described in detail in Scott et al [28].…”

An oxocarbenium-ion intermediate of a ribozyme reaction indicated by kinetic isotope effects

“…PRPP was prepared from isotopically derivatized D-ribose, either 1-2 H (98%) or 1-13 C (99%) substituted from Cambridge Isotope Laboratories (Cambridge, MA). Ribose 5-phosphate was synthesized from ATP and ribose by using ribokinase (EC 2.7.1.15), and pyrophosphate was transferred from ATP by using phosphoribosyltranferase (EC 2.7.6.1) to generate PRPP (17,18). Ribose 5-phosphate and PRPP were purified on a DEAE A-25 column, with a linear gradient from 50 to 900 mM triethylammonium bicarbonate (TEAB), pH 7.0.…”

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].…”