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'

In the paper by Azad and co-workers, the authors provide a comprehensive protocol for the synthesis of specifically deuterated dNTPs. This very throrough protocol, along with a validation on a small RNA motif will provide the community with new tools to study RNA structure and dynamics using both NMR and OH radical footprinting. The protocol builds on existing synthetic approaches adding significant functionality to the strategy and should be published. I have several comments the authors should consider in a revision to clarify and expand on their work.

1.) The authors demonstrate the value of specifically deuterating dNTPs by performing OH footprinting experiments and establishing a mechanism for this common structural probe. They also mention how such dNTPs can be used to study RNA dynamics using NMR. Nonetheless I think there may be a much broader use for this strategy and I believe the manuscript would be strengthened by a more up front discussion (in the introduction even) of how such dNTPs could be used. I would encourage the authors to for example speculate on how specifcally deuterated dNTPs might affect sequencing in a nanopore (e.g. the minIon system) or even on the Pacific Biosystems sequencer. I think if the authors can propose a use (even hypothetical) for their specifically labeled dNTPs using next generation sequencing, the potential breadth of their protocol could be increased.

2.) The protocol is well described and seems accessible to a broad audience, that being said for a non-chemistry lab it is still daunting. One thing that I find useful in other "protocol" journals is a highlighting of "key" or important steps. In particular notes about quality control steps that can help "debug" the protocol if it is not working. As such I think the protocl could be expanded some with such useful tidbits of practical information, the authors could inspire themselves from the format in Nature Protocols for example.

3.) Do the authors have any plans for commercializing these dNTPs? The authors may wish to have a section explaining under what circumstances they may be willing to share some refined materials with other labs. In practice, if I wanted to use such protocols I would probably first e-mail the corresponding author to ask for an aliquot to see if the experiment I wish to carry out will work. Then, if the results are promising I would start the synthesis. As such, I expect the authors to get many requests for these materials when the protocol is published, and I would encourage them to disclose under what conditions they may be willing to share the synthesized materials.

4.) The authors may wish to cite and mention this protocol (http://www.sciencedirect.com/science/article/pii/S0076687909680034) for using SAFA, which includes video tutorials in case users are not familiar with the software.