I posted it there mainly out of principle (openness is good), but it's already attracting some critical commentary. This reminded me that one of the main purposes of the arXiv is to encourage pre-publication discussion of research. This is open peer review!
So please post your comments on our manuscript here. To get things started, here are the comments already made:
NotAnAstrobiologistJan 31, 2012 09:42 PM
As I understand it, Figure S1 has error bars which represent the standard deviation of ion counts for independent purifications of the same DNA sample, characterizing the variance across purifications.Later:
Why use the standard deviation in this case where your sample size=2? Using the two actual values would make more sense to me (estimating the distribution in this case obfuscates the underlying data, as you've irreversibly "reduced" two observed values to two statistical estimates). I think it makes more sense to show the actual observations, or do (at least) three experiments...
FWIW to make sure I wasn't making it up (I've seen error bars on small n estimates before), note the line:
"However, if n is very small (for example n = 3), rather than showing error bars and statistics, it is better to simply plot the individual data points."
Error bars in experimental biology
http://jcb.rupress.org/content/177/1/7.full
Or have the error bars indicate the range rather than standard deviation?
I don't want to be the person suggesting new experiments, but Michael Bartlett's comments about ICP-MS (in the recent C&E News article) *sound* kind of nice. As far as I can tell ICP-MS would not rely on the assumption that AsDNA being hydrolyzed. You've obviously been very careful to state where that assumption is present.
ReplyDeleteI understand the cost/return ratio here, but if it is as easy as sending a few small samples to someone (delaying the paper by a few weeks, say)...it might be worth it.
Does someone who is more of an expert in MS care to comment on how much more would ICP-MS buy in terms of conclusive strength?
One possible objection to the LC-MS data is that they depend on sufficiently complete DNA digestion. I don't see anywhere in the manuscript where you confirmed complete digestion. You cite a published reference that your method is based on, but that reference actually used P1 followed by alkaline phosphatase, rather than P1 followed by snake venom exonuclease as you did.
ReplyDeleteP1 is supposedly not active on dsDNA, and you don't indicate that you denatured your DNA before digestion. So the majority of your digestion might have come from the 3' exo activity of the venom nuclease.
Imagine a random dsDNA fragment with occasional arsenates. If arsenate diesters are resistant to digestion (very possible), then the exo would digest from each free 3' end up to the first arsenate linkage and stop. If P1 remains active after you adjusted the buffer for the exo treatment, then P1 would eliminate the resulting 5' overhangs. That would give you plenty of 5'-mononucleotides to be detected in your LC-MS. However, it would also leave you with the remaining dsDNA fragment with an arsenate linkage at each 3' end (and possibly internally as well). Such fragments would probably be much too large to show up as well-resolved extra peaks in the LC chromatogram.
I admit this is a bit contrived, but it might be worth adding a line somewhere to at least indicate that you know the digestions really were complete. (Or, if you don't actually know that, you could quickly do another set of digestions on the different DNAs and run them on gels to confirm there are no remaining fragments of any significant size. If it was me, I'd probably run some on agarose to confirm that there's nothing really large, and some on PAGE to look for small stuff.)
One other minor suggestion. I don't think you should use the term "dideoxy" the way you have. Dideoxy typically refers to nucleosides or nucleotides that lack hydroxyls at both 2' and 3' position (e.g. the chain terminators used in Sanger sequencing). I think the more correct term would be dinucleotide monophosphates (or mono-arsenates), or simply dinucleotides.
What was the As containing standard in LS/MS? Was it the same compound that you were trying to quantify in digests?
ReplyDeleteAnonymous,
ReplyDeleteThe posts below show the Rosie was looking for free arsenate or heavy nucleotides. She ran a positive control on the arsenate.
If I interpret Table 1 correctly, the LCMS identifies the (expected) existence of dAMA and dAMA-dAMP. It's a little tangential, but are the detected amounts of these compounds in line with expected values?
It is common sense and textbook knowledge that Arsenic cannot replace Phosphorus in its esters and survive even minimal contact with water! NASA’s paper was a disturbing perversion of science by some shameless and uneducated pseudo-researchers looking for cheap thrills and immediate sensations.
ReplyDeleteI would like to thank the authors of the manuscript for rebutting the frivolous claims.
Rosie's new manuscript should be considered as a confirmation of decades of solid scientific research that demonstrate that Arsenic cannot replace Phosphorus. The Occam's razor principle should be enough to dispel convoluted alternative explanations like Ross Durland's.
ReplyDeleteCome on, anonymi, sign your names...
ReplyDeleteLest anyone should be confused, I don't believe there really is any As in the GFAJ-1 DNA. But there's no point publishing the manuscript unless the experiments and data are robust. Regardless of whether my specific convoluted scenario is plausible, I think that incomplete digestion would render the LC-MS data less conclusive. Simce that data is the primary experimental basis for rejecting the claim of As-DNA, I think evidence of complete digestion is important.
ReplyDeleteAll IMHO, of course.
Ross,
ReplyDeleteYour point is understood, and a good one to make, but we were more careful than you give us credit for, and there are a number of inaccuracies regarding your statements about digestion. For instance, Wang et al also used snake venom exonuclease following pH adjustment.
"Samples (50 μg in 200 μL) of isolated DNA or oligodeoxynucleotide were hydrolyzed with 4 U nuclease P1 (USBiological) in 0.3 M sodium acetate, pH 5.6, 0.5 mM ZnCl2, 0.5 mM deferoxamine mesylate (Sigma) at 37 °C for 2 h, as described in previous studies of DNA damage analysis.5 Following adjustment of pH (200 μL of 30 mM sodium acetate, pH 8.1), 40 U of alkaline phosphatase (New England Biolabs) and 2 U of snake venom phosphodiesterase (Crotalus adamanteus, USB) were added and the mixture was incubated at 37 °C for 6 h before centrifugal ultrafiltration using Microcon YM-10 (Millipore)."
It wasn't stated in the paper, but it is easy to know whether the digestion was complete by measuring the A260 of the filtered-concentrate (3 kDa cutoff) post-digestion. This data was not presented, but we could add a sentence in the methods indicating explicitly why we know the gDNA was digested to completion. Although admittedly slightly cryptic, the "incomplete digest" of figure 4C/D does imply that complete digestion was measured.
Given existing literature about arsenate-esters, there are no reasons for anyone to believe that a hanging 3'-arsenate-nucleotide wouldn't degrade to arsenate and a nucleoside within seconds once rest of the DNA is digested.
Finally, I think you're right about the confusing-ness of the nomenclature. We should probably change it review.
Marshall,
ReplyDeleteThanks for the clarification. I guess I didn't read that reference closely enough. I agree that if there was no residual A260 material in your retentates, that's a strong indication of complete digestion.
And just to be clear, I never meant to imply that you didn't check for complete digestion. Only that it wasn't stated in the manuscript. If It seemed otherwise, I certainly apologize.
I agree that a terminal 3' arsenate diester should readily hydrolyze. But you've already entertained the possibility that arsenate diesters are somehow much more stable in DNA. Otherwise there would be no reason for LC-MS at all, right? So yes, if it were up to me, I would still want to see a sentence added to describe how you confirmed complete digestion.
"Given existing literature about arsenate-esters, there are no reasons for anyone to believe that a hanging 3'-arsenate-nucleotide wouldn't degrade to arsenate and a nucleoside within seconds once rest of the DNA is digested."
ReplyDeleteThis statement is untrue. The MONOester undergoes hydrolysis with a half life of 30-40 minutes, not < a second as you suggest (of course this is not true for the diester linkage that would be required to produce DNA polymers, which does have extremely short half-life). This timescale is completely sufficient to allow for preparation and characterization of dAMA, as was done nearly 30 years ago in (1).
Thus, the assumption that free arsenate is an acceptable proxy for any arsenate that once existed in mononucleotides is dependent on the amount of time that took place between enzymatic production of these mononucleotides and your MS analysis. So for example, if we grant FWS her 4% incorporation and produce mononucleotides from her DNA, how much of that 4% we would be able to detect by hydrolysis to arsenate is ~0% if no time elapsed between enzymatic treatment and MS, ~2% if 40' took place between enzymatic treatment and MS, and so on and so forth.
Of course, the simple remedy to this solution is to look for the mononucleotides as well and verify that there are no dAMAs present in the sample. You state in your manuscript
""We also did not detect any arsenate-conjugated mono- or dinucleotides by exact mass (Figures
4B and 4D). Although no standards are available for these molecules to validate retention time
and ionization efficiency..."
While I appreciate that if a dAMP flies well we might like to think that a dAMA flies equally well, without a standard showing us that that is true why should we believe you? The reply that "no standards are available" is false— maybe you can't buy them from Sigma but people produced and characterized these materials more than 30 years ago, I dont think its asking too much of you guys to do this as well.
Alternatively, if enough half-lives elapse between enzymatic treatment and your MS analysis (waiting 24 hours would probably be satisfactory to be >99% hydrolyzed using the values from (1)).
(1): Arsenic mononucleotides. Separation by high-performance liquid chromatography and identification with myokinase and adenylate deaminase
Rosario Lagunas, David Pestana, and Jose C. Diez-Masa
Biochemistry 1984 23 (5), 955-960
Marshall,
ReplyDeleteI took another look at the Wang et al reference you cite from PNAS 2011. I don't see that passage you quoted. Is it possible you meant to cite an earlier paper by Wang? I see there was one In Nature Chem Biol 2007 on the same topic, but I don't have full text access.
I would like to see a 260/280 ratio or an absorbance spectrum on the DNA used in experiments. (Criticism of this and FWS)
ReplyDeleteThought about remaining As on DNA:
There may be tight-binding arsenate-binding transcription factors hanging on.