[guest post: Alex Bradley, PhD] Arsenate-based DNA: a big idea with big holes

In the wake of the NASA excitement over the new arsenic study, and my promise to give a detailed review of the paper itself, I have recruited a colleague with strong opinons about the work, a solid chemistry and microbiology background, and "Dr." in front of his name to share his analysis. I will be posting have posted my personal and less-technical take on the whole thing soon, so stay tuned as well. Dr. Alex Bradley uses modern geochemistry and microbiology tools to study the evolution of life and Earth. He has the following to say about the paper.

ResearchBlogging.org

There's been a lot of hype around the news of GFAJ-1, the microbe claimed to substitute arsenate for phosphate in its DNA. In the midst of all the excitement, one thing has been overlooked:

The claim is almost certainly wrong.

The study published in Science has a number of flaws. In particular, one subtle but critical piece of evidence has been overlooked, and it demonstrates that the DNA in question actually has a phosphate - not an arsenate -backbone.

To understand why, we need to back up a bit. One thing that everyone agrees on is that all things being equal, DNA with an arsenate backbone will hydrolyze quickly in water, while DNA with a phosphate backbone will not. Steve Benner has pointed out that the half-life of the hydrolysis reaction is about 10 minutes.

Wolfe-Simon et al. recognize this, but claim that the bacterium GFAJ-1 must have some unknown biological mechanism to compensate, and this prevents the DNA from falling apart in the cells. Let's assume for now that they are correct. It might be plausible - biology has all kinds of strange tricks and this idea can't be quickly dismissed, even if it seems radical.

But chemistry is much more predictable. Once DNA is out of the cell, pure chemical processes take over, and experiments have demonstrated that hydrolysis of arsenate links is fast. So you could do a simple experiment to test whether DNA had a phosphate or arsenate backbone: just remove DNA from the cell and put it in water for a few minutes. Then examine whether it hydrolyzes or not.

In an accidental way, Wolfe-Simon et al. performed precisely this experiment. The result indicates that the DNA of GFAJ-1 has a phosphate backbone.

The details are these: to isolate DNA, Wolfe-Simon et al. performed a phenol-chloroform extraction. In this technique, after cellular disruption, DNA and other cellular material were dissolved in water, and then the non-DNA material (such as lipids and proteins) were cleaned out of the mixture using phenol and chloroform. This is a pretty common laboratory procedure, and typically would take an hour or two. But here is the key point:

During this whole procedure, the DNA was in water.

Remember, proteins were removed from this mixture. Any cellular machinery that stabilized arsenate-DNA was removed. In the absence of biochemistry, pure chemistry takes over: any arsenate-DNA would have been quickly hydrolyzed in the water, breaking down into fragments of small size. Alternatively, phosphate-DNA would not hydrolyze quickly, and large-sized fragments might be recoverable.

So what size are the fragments of DNA extracted from GFAJ-1? They are large. Figure 1 shows a single strong band. This pattern is a bit unusual for a genomic DNA extract, but the important thing is that the fragments in this band have around 10,000 nucleotides between breaks in the DNA. These long chains of nucleotides did not hydrolyze in water. Yet it is precisely this DNA band that is claimed to have an arsenate backbone.

How can this be?

The answer is: it can't be. If this DNA did not hydrolyze in water during the long extraction process, then it doesn't have an arsenate backbone. It has a phosphate backbone. It is normal DNA.

So what accounts for the claim of arsenic in this DNA? Wolfe-Simon et al. used a technique called nanoSIMS to analyze elemental concentrations of the agarose gel at the location of the DNA band. They determined that the part of the gel containing DNA also contained both arsenic and phosphorus. But what did they really analyze?

The answer is that the nanoSIMS determined the concentration of arsenic in the gel - not specifically in the DNA. Arsenic was present in the gel at the location of the DNA band. But these data do not require that arsenic is part of the DNA, only that it is somehow associated with the DNA. So here is a more plausible explanation: arsenate sticks to stuff. When you grow bacteria in media containing lots of arsenate, cellular material gets covered in arsenate. If you analyze this material chemically, you see a high arsenic background. The arsenic background will remain even after you separate the cellular material into its constituent parts - DNA, lipids, and proteins - because the chemical separation is imperfect. You could imagine a parallel experiment: if you grew bacteria in seawater, a band of DNA extracted from these bacteria might show a high background of sodium and chloride. This would not be very surprising - and it certainly wouldn't imply that the DNA had a chloride backbone.

Wolfe-Simon and her colleagues might quibble with this, and claim that arsenate is not that 'sticky'. This should have been resolved by running a negative control. Grow some bacteria with phosphate-backboned DNA in media containing high concentrations of arsenate. Then extract the DNA, run a gel, and just demonstrate that the gel does not have a high arsenic concentration associated with the DNA band. That would be evidence that my explanation is wrong. But this simple control was not performed in study published in Science.

One objection to my claim might be: if the GFAJ-1 DNA contains phosphate, where did the phosphate come from? The researchers claim that there wasn't much phosphate in their growth media. In fact, they did a very good job of quantifying the background phosphate concentration: it was about 3 micromolar, which was certainly much lower than the arsenate concentrations (by a factor of about 10,000).

But here's the relevant question: Is 3 micromolar phosphate a lot? Or a little? One point of comparison is the Sargasso Sea, where plenty of microbes survive and make normal DNA. Here, the phosphate concentrations are less than 10 nanomolar - or 300 times less phosphate than the "phosphate-free" media in the GFAJ-1 experiment. At such low phosphate concentrations, some bacteria compensate by removing phosphorus from their lipids - but not from their DNA.

So the Sargasso Sea tells us that some bacteria are capable of making DNA at very low phosphate concentrations. The most plausible explanation is that the bacterium GFAJ-1 can make normal DNA at micromolar phosphate concentrations, and that it also has the ability to tolerate very high arsenate concentrations.

There are numerous other aspects of this study that don't make sense. Why would bacteria from Mono Lake need the ability to substitute arsenate for phosphate in their DNA? Yes, arsenic concentrations are high in Mono Lake. But so are phosphate concentrations, which approach 1 millimolar - or 100,000 times higher than in the Sargasso Sea. Mono Lake has more phosphate available than nearly any other environment on Earth. There is no selective pressure for the evolution of what would surely be a massively complex switch in nucleic acid chemistry from phosphate to arsenate. I can only begin to imagine the structural problems that would be imposed on DNA by this switch, which would change bond lengths between nucleotides, and cause secondary problems with transcription, etc. Then there is the radical suggestion that nucleotide chemistry is stable because might occur in a 'non-aqueous' environment. It's not clear how that could work.

Finally, there's a simple experiment that could resolve this debate: analyze the nucleotides directly. Show a mass spectrum of DNA sequences demonstrating that nucleotides contain arsenate instead of phosphate. This is a very simple experiment, and would be quite convincing - but it has not been performed.

This study lacks any real evidence for arsenate-based DNA; unfortunately these exciting claims are very very shaky.

Update (12/6/2010): Dr. Rosie Redfield has a quantitative discussion of why there's plenty of phosphorus here.

Wolfe-Simon F, Blum JS, Kulp TR, Gordon GW, Hoeft SE, Pett-Ridge J, Stolz JF, Webb SM, Weber PK, Davies PC, Anbar AD, & Oremland RS (2010). A Bacterium That Can Grow by Using Arsenic Instead of Phosphorus. Science (New York, N.Y.)

PMID: 21127214

 

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