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Review of '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'

Extends and refines the method developed by Tolbert and Williamson
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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

 Robert Azad,  Shakti Ingle,  Tom Tullius (corresponding) (2015)
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.

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    Azad et al. present a well-written and detailed protocol for the synthesis of ribonucleotide triphosphates with the ribose site-specifically labeled with deuterium. This work extends and refines the method developed by Tolbert and Williamson for nucleotides with multiple deuterium atoms. The authors then illustrate an application of the NTPs with the synthesis of an oligonucleotide for hydroxyl radical cleavage studies. Though daunting, the protocol is comprehensive in describing sources of materials and supplies, preparation of enzymes, NTP synthesis and NTP purification. The level of methodological detail appears to be high enough for a relatively inexperienced researcher to reproduce the results and the authors have pointed out critical steps, idiosyncrasies and nuances throughout. The chromatographic purification steps are especially helpful given the difficulties of NTP purification and working with ion-pair and boronate chromatography techniques. The work will prove useful to the nucleic acid community. The on-line format will allow researchers to update with their own experience, correct weaknesses, and provide additional methodological insights. A few minor comments and suggestions:

    • There are many different applications for isotopically-labeled ribonucleotides beyond hydroxyl radical kinetic isotope effect studies, such as site-specific or uniform labeling with 13C and 15N for mass spectrometric studies of oligonucleotides and tRNA sequences. Since NTPs with site-specific labels or labeling patterns are not always commercially available, the method described in this paper could very easily be adapted as a general approach to synthesizing isotopically labeled ribonucleotides, as well as ribonucleosides by dephosphorylation, starting with labeled bases and ribose. A discussion about potential alternative applications would broaden the impact of the protocol. Perhaps simply changing the title to something like “Isotopically-labeled nucleotides as chemical probes of RNA structure:….” would generalize the method while keeping an immediate focus on the deuterium labeling application.

    • Given the potential need for repeat syntheses on a major project, some comment about experience with stability during storage for the NTPs and enzymes would be helpful, perhaps noted at appropriate steps in the protocol.

    • It would be useful to have a discussion of trouble-shooting, frequently occurring problems, pitfalls and alternative strategies in a section at the end of the protocol. One of the benefits of ScienceOpen will be to have commentary added by other researchers, but some discussion of known problem points would benefit the inexperienced researcher.

    • Perhaps I missed it, but an estimate of the time required for each major step would be helpful in planning a project. 


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