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A, T, C, G, X and Y: An New Organism with Unnatural DNA

by Ygggdrasil
Tags: synthetic biology
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Ygggdrasil
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May7-14, 01:22 PM
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Published today in Nature, researchers report engineering an organism capable of replicating DNA with an unnatural base pair (X-Y) in addition to the two normal base pairs found in DNA (A-T and C-G).

Here's what the unnatural base pair looks like (in comparison to the C-G base pair):


Here's the abstract for the article:
Organisms are defined by the information encoded in their genomes, and since the origin of life this information has been encoded using a two-base-pair genetic alphabet (A–T and G–C). In vitro, the alphabet has been expanded to include several unnatural base pairs (UBPs). We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS–dNaM), which is efficiently PCR-amplified and transcribed in vitro, and whose unique mechanism of replication has been characterized. However, expansion of an organism’s genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must be available inside the cell; endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA containing the UBP within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into Escherichia coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid containing d5SICS–dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a notable growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to propagate stably an expanded genetic alphabet.
Malyshev et al. A semi-synthetic organism with an expanded genetic alphabet. Nature. Published online 07 May 2014. doi:10.1038/nature13314.

It'll be interesting to see what (if any) applications come from this work. It seems like there's still some work to be done in order to make it a little easier for these organisms to naturally use the new bases (for example, maybe getting the organisms to synthesize them by themselves).
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Greg Bernhardt
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May7-14, 07:22 PM
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Lovely news! I was going to post this story too!

Here is the layman version
http://www.huffingtonpost.com/2014/0...n_5283095.html

Potential uses of the technology include the incorporation of a toxic amino acid into a protein to ensure that it kills only cancer cells, and the development of glowing amino acids that could help scientists to track biological reactions under the microscope. Romesberg’s team has founded a company called Synthorx in San Diego, California, to commercialize the work.
Astronuc
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May7-14, 11:37 PM
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I heard this today, and I could only think of the ramifications of it not working as expected. It seems to work in the lab, in a limited system. I'm not sure it would necessarily work as expected, especially if it mutates.

I can see this being the basis of a great science fiction story in which a 6-letter [superior] lifeform takes over and attempts to replace the 4-letter varieties.

Yanick
#4
May8-14, 08:13 AM
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A, T, C, G, X and Y: An New Organism with Unnatural DNA

I'm no expert in this area to be sure but, I think this work is a far cry from the Huffington Post's statements about applications of the technology. Not to take anything away from the work, it is definitely interesting but translation of these unnatural bases into proteins will likely take a long time. I don't have access to the full paper, but I would like to see their data and really read what the authors have achieved.

Unnatural amino acids can already be incorporated into proteins, see the work of Peter Schultz at Scripps: http://schultz.scripps.edu/publications.php?500
Pythagorean
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May8-14, 09:15 AM
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Quote Quote by Astronuc View Post
I heard this today, and I could only think of the ramifications of it not working as expected. It seems to work in the lab, in a limited system. I'm not sure it would necessarily work the expected, especially if it mutates.

I can see this being the basis of a great science fiction story in which a 6-letter [superior] lifeform takes over and attempts to replace the 4-letter varieties.
I think the general idea is it takes so much work to introduce just one small change without compromising the integrity of the whole system that it's highly improbable that such fears will ever be realized. A general knowledge of evolution might lead you to believe that one mutation can lead to huge catastrophic changes, but the big changes are the result of many mutations over a long time scale.

We often read about how one small change allowed for larger brains in humans (like the reduction in jaw muscle size) and while it's probably true that we wouldn't have big brains without that mutation, there were so many more mutations and so much more time beyond that event that were required.
Ygggdrasil
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May8-14, 09:39 AM
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Quote Quote by Yanick View Post
I'm no expert in this area to be sure but, I think this work is a far cry from the Huffington Post's statements about applications of the technology. Not to take anything away from the work, it is definitely interesting but translation of these unnatural bases into proteins will likely take a long time. I don't have access to the full paper, but I would like to see their data and really read what the authors have achieved.
All of the popular press reports of the study take it way past any data that the authors show in the paper. Introducing unnatural amino acids into bacteria is certainly a potential application of the work (although I'm not sure how they come up with the 172 amino acids number), but the authors present no data regarding this application. In fact, I'm not aware of any study showing that the ribosome can decode unnatural base pairs (though if someone knows of a study, please let me know). Also, other, potentially better, strategies exist for expanding the genetic code to use multiple unnatural amino acids, such as a ribosome engineered to read 4-base codons, rather than 3-base codons (http://www.nature.com/nature/journal...ture08817.html).

The paper is a nice proof of principle that an unnatural base pair can replicate within bacteria, which is an important step because it shows that these unnatural base pairs can persist in living cells without being recognized and removed by DNA repair enzymes (a worry scientists had prior to this study).

As someone who studies protein-nucleic acid interactions, I'm most excited about the potential applications where we use the unnatural base pair to introduce a label (such as a fluorophore or a photoreactive group for crosslinking to binding partners) at a specific location in DNA or RNA in living cells. This is difficult to do with current technologies, but if this technology makes it possible, it could enable some really neat experiments.

For those worried about this organism somehow escaping and causing havoc in the world, it won't. Because the organism cannot synthesize the unnatural nucleotides by itself, it cannot use the unnatural base pair unless you actually provide it with the unnatural nucleotides. Because these nucleotides do not exist in nature outside of the lab, if this bacteria would escape, it would not make any unnatural DNA.
Andy Resnick
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May8-14, 12:04 PM
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Quote Quote by Ygggdrasil View Post
Published today in Nature, researchers report engineering an organism capable of replicating DNA with an unnatural base pair (X-Y) in addition to the two normal base pairs found in DNA (A-T and C-G).

<snip>
Whoa... reading the paper now. very cool.
cryora
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May10-14, 11:16 AM
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I'm perplexed by the 172 amino acid number as well. For that to be possible, it would require new tRNA's with anticodons that recognize the newnucleic acids, and new aminoacyl-tRNA synthetases to catylize bonds between the respective amino amino acids and the new tRNA (just going off of what I learned in my Bio 101 class). Unless, the cell was able to incorporate the existing system to do the nucleotide-amino acid conversion. The huffington post article did say that as soon as it ran out of the new nucleotides, it started using regular ones in place of them, which I find funny. The only thing I can deduce from the introduction of two new nucleotides is that, assuming ribosomes continue to read 3 letter codons at a time, then the possible number of codons with 6 possible nucleotides instead of 4 is 6^3 = 216 different combinations of codons instead of 4^3 = 64.
mfb
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May10-14, 11:28 AM
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172: There are 216-64 = 152 new triplets to add up to 152 new amino acids (at least in principle) to the 20 existing ones without having to re-write the existing code.
Murdstone
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May31-14, 10:52 AM
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Thanks Ygggdrasil for bringing this topic up for review. I do and don't understand the information.

What is the uniqueness of X and Y? Is it that chemically they efficiently bind X between A and C and Y between T and G without upsetting the integrity of the original structure?

What does this mean - "the unnatural nucleoside triphosphates must be available inside the cell"?

Is the bacteria actually undergoing mitosis?
Ygggdrasil
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May31-14, 12:23 PM
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Quote Quote by Murdstone View Post
What is the uniqueness of X and Y? Is it that chemically they efficiently bind X between A and C and Y between T and G without upsetting the integrity of the original structure?
DNA consists of a double helix held together by interactions between the bases in DNA. In normal DNA, A will pair with T across the double helix and C will pair with G. More importantly, the nucleotides mispair (e.g. C sits across from T) at extremely low frequency, which underlies the ability for DNA to store information, replicate that information during cell division, and to copy that information into RNA.

What researchers have done is to create a new pair of bases that adds a third type of base pair to the existing A-T and C-G base pairs. This new can exist in a normal DNA double helix without significantly disrupting its structure. This in theory allows the DNA to store more information because the DNA now encodes six bits (A,T,G,C,X,Y) instead of the normal four bits in natural DNA (A,T,C,G).

What does this mean - "the unnatural nucleoside triphosphates must be available inside the cell"?
Cells do not normally make the X and Y nucleotides, so in order to get bacteria to replicate DNA containing the unnatural base pair, the researchers had feed the pre-made nucleotides to the bacteria. In order for this to be successful, the researchers first had to engineer these bacteria to make a protein they isolated from algae that allows the bacteria to transport the unnatural nucleotides into the cell.

Is the bacteria actually undergoing mitosis?
Mitosis is the specialized process by which eukaryotic cells undergo cell division. The researchers worked with bacteria, and bacteria do not undergo mitosis. While bacteria undergo cell division, the process is much less complicated than in eukaryotes. But yes, the authors do show that the unnatural base pair gets copied during cell division.


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