Abstract
Abstract We describe here a detailed protocol for the synthesis of ribonucleotides specifically deuterated at each ribose carbon atom. We synthesized 20 specifically deuterated ribonucleotides: ATP, CTP, GTP, and UTP, each of which contained one of five deuterated riboses (either 1′-D, 2″-D, 3′-D, 4′-D, or 5′,5″-D2). Our synthetic approach is inspired by the pioneering work of Tolbert and Williamson, who developed a method for the convenient one-pot enzymatic synthesis of nucleotides (Tolbert, T. J. and Williamson, J. R. (1996) J. Am. Chem. Soc. 118, 7929–7940). Our protocol consists of a comprehensive list of required chemical and enzymatic reagents and equipment, detailed procedures for enzymatic assays and nucleotide synthesis, and chromatographic procedures for purification of deuterated nucleotides. As an example of the utility of specifically deuterated nucleotides, we used them to synthesize specifically deuterated sarcin/ricin loop (SRL) RNA and measured the deuterium kinetic isotope effect on hydroxyl radical cleavage of the SRL.
Highlights
Nucleoside 5′-triphosphates (NTPs) in which the ribose is deuterated are valuable in structural and biochemical studies of nucleic acids
A breakthrough in making deuterated nucleotides more widely available came from Williamson and coworkers, who developed an enzymatic approach for the synthesis of deuterated ribonucleoside 5′-triphosphates from isotopically labeled glycerol or glucose [5,6,7]
We describe here a comprehensive protocol, based on Williamson’s pioneering work [6, 7], for the one-pot enzymatic synthesis and purification of milligram quantities of deuterated adenosine 5′-triphosphate (ATP), guanosine 5′-triphosphate (GTP), uracil 5′-triphosphate (UTP), and cytidine 5′-triphosphate (CTP), for use in chemical probe experiments on RNA structure
Summary
Nucleoside 5′-triphosphates (NTPs) in which the ribose is deuterated are valuable in structural and biochemical studies of nucleic acids. A breakthrough in making deuterated nucleotides more widely available came from Williamson and coworkers, who developed an enzymatic approach for the synthesis of deuterated ribonucleoside 5′-triphosphates from isotopically labeled glycerol or glucose [5,6,7] Their scheme was able to produce milligram quantities of NTPs sufficient for preparing RNA by in vitro transcription for use in NMR spectroscopy. Substantial quantities of ATP, GTP, unreacted UTP, and ADP are present in the final CTP synthesis reaction mixture As we developed this method, a balance had to be struck between the high resolving power of HPLC and the constraint on column capacity inherent in using an analytical-scale instrument. The analysis reveals a substantial reduction in cleavage only at nucleotides that contain deuterium, indicating that RNA strand cleavage can be initiated by abstraction of a C5′ hydrogen atom from the ribose backbone
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