Deuterated nucleotides as chemical probes of RNA structure: a detailed protocol for the enzymatic synthesis of a complete set of nucleotides specifically deuterated at ribose carbons

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. INTRODUCTION Nucleoside 5′-triphosphates (NTPs) in which the ribose is specifically deuterated are valuable in structural and biochemical studies of nucleic acids. They can be used, for example, to reduce the complexity of NMR spectra [1] and to discern mechanistic details of nucleic acid cleavage [2]. Such studies provide information not otherwise obtainable. For example, we used specifically deuterated deoxynucleotides to probe the mechanism of DNA cleavage by the hydroxyl radical [3]. The ability to chemically synthesize specifically deuterated DNA allowed us to determine quantitatively which hydrogen atoms of deoxyribose are abstracted by the hydroxyl radical from duplex DNA. This work both elucidated the chemical and structural mechanism of a widely used chemical footprinting agent and contributed to a more detailed understanding of ionizing radiation-induced DNA damage. Despite wide interest in the results of this work, the methods we described have seldom been used because of the difficulty of producing specifically deuterated NTPs by chemical synthesis [4]. Chemical routes to NTP synthesis require multiple steps that often are laborious and time-consuming. The consequence is that specifically deuterated nucleotides have been largely inaccessible to the structural and molecular biologists who would be most interested in using them. A breakthrough in making specifically 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 [5–7]. Their scheme was able to produce milligram quantities of NTPs sufficient for preparing RNA by in vitro transcription for use in NMR spectroscopy. They showed that the enzymes did not have to be highly purified to be effective, which significantly reduced the effort and cost of NTP synthesis. Their synthesis is convenient, as NTPs are produced in a “one-pot” reaction that involves multiple enzymes. Williamson and coworkers used this approach to produce NTPs with multiply deuterated ribose residues, because they used commercially available uniformly deuterated glucose or glycerol in their synthetic protocols [6, 7]. While multiply deuterated NTPs are useful for NMR experiments, our earlier work [3] and the work of others [2] showed that selective deuteration at a specific deoxyribose carbon SOR-LIFE