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Artificial Base Pairs in Living Cells

Synthetic biology seems to have taken another big step. Many labs over the years have tried out expanding the genetic code in various ways, but all these run in various in vitro systems. Now the first organism has been engineered with a working unnatural base pair, according to this paper in Nature from the Romesburg group at Scripps.
The base pair in question is d5SICS and dNaM, shown at left, and a history of how they were developed is here. This class of interaction was found by screening thousands of possible combinations, and it’s notable that there’s no hydrogen bonding going on between the two residues. (It’s worth keeping in mind that the current AT/CG base pairing system was presumably also arrived at by screening a wide variety of candidates until something worked!)
There are a number of tricky steps needed to get this to work:

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.

A transporter spliced in from algae can bring in the unnatural triphosphates, as it turns out, but a next step would be getting enzymatic machinery inside the cell to make them. But the existing enzymes can handle them once they’re available, and replicate plasmids containing these pairs, which also don’t get tagged as DNA errors and snipped out by any of the endogenous repair mechanisms. So another bridge has indeed been crossed.
Romesburg has started a company, Synthorx, to try to take advantage of the chemical biology possibilities in this work. (I realize that I’m probably supposed to think “Syntho-Rx” when I see that, but my brain persists in saying “Syn-thorks”.) I can imagine, down the road, some very interesting assay development possibilities that follow from this technique, with what might be very high signal/noise ratios, so this is worth keeping an eye on.
Update: a criticism of the press coverage of this paper, which has indeed not been very well informed.

10 comments on “Artificial Base Pairs in Living Cells”

  1. Eric Sprague says:

    I read “Synthorx” in connection to something like this, and immediately think of “synthetic orcs”, which we don’t need 🙂

  2. Denris says:

    I read it as “Synth rocks!”, which it does.

  3. I don't understand says:

    From the lab site:
    “Remarkably, we have found that neither H-bonding nor large aromatic surface area is required for base pair stability in duplex DNA or polymerase mediated replication.”
    As far as I know, Arthur Kornberg (or somebody else in Stanford) has already proven that hydrogen bonding is not the reason for the polymerase to work. As long as the nucleotides fit in the pocket, polymerase will be happy to move on to the next.
    “From studies done with nonpolar isosteric analogs of natural DNA bases which lack the ability to form Watson-Crick (W-C) hydrogen (H) bonds, it has been concluded that W-C H bonding is not needed for DNA synthesis by classical high-fidelity DNA polymerases (30, 32, 33); rather, the geometric fit of the incoming nucleotide with the templating base within the polymerase active site is the principal determinant of the efficiency and fidelity of DNA synthesis in these polymerases (7, 9, 22).”
    I understand the importance of their work but I can’t understand why this made such a great impact in the last 24 hours.

  4. Wavefunction says:

    #3: That was Eric Kool from Stanford. I still remember reading that paper as an undergraduate and staring at it with amazement. His study as well as this one tells us just how adaptable nature can be.

  5. elementary says:

    I agree the media seems to be arbitrarily running away with this research article (I guess being in Nature helps). The “firsts” in taglines and news report titles are also being thrown around haphazardly. Kool has shown artificial DNA bases functioning (at least read through with fidelity) and not being repaired in living organisms.
    Yes Romesberg took it further with the triphosphate incorporation which is pretty neat, but there’s still a ways to go here.

  6. Dr. Manhattan says:

    There i indeed still a ways to go! A lot of the press releases are probably aimed at generating VC interest in the new company. As long as there are VCs out there long on cash and short of true understanding of the complexities of biological technology, there will always be these kind of triumphant declarations; the purpose being to raise money.

  7. Triazine says:

    Having to externally supply the base pairs would be an effective way to prevent fertile genetically modified organisms from escaping into the wild. InGen should have explored this method, rather than their disastrous lysine contingency plan.

  8. David Borhani says:

    Cool, but agree there’s a long way to go: Just to lay out some of those needed steps:
    1. Biosynthesis of the unnatural bases. I suspect there’s a reason why we have uracil/thymine, cytosine, adenine, and guanine (check out how cells make them…some pretty nice “organic” chemistry).
    2. Connection of base to deoxy/ribose (e.g., via purine nucleoside phosphorylase, phosphoribosyltransferases, etc).
    3. Monophosphorylation — deoxycytidine kinase and the like. Key step where many would-be therapeutic nucleoside analogues fall down. Authors note that this seems to work for analogues of the new bases described here.
    4. Triphosphorylation — Nucleoside-diphosphate kinase. Authors note less satisfactory results at this step — hence their approach to supply unnatural NTPs w/ an NTP transporter.
    Also, anyone know about the protein *translation* capabilities of these new nucleotides? Are there “orthogonal” tRNAs/tRNA synthetases already developed for them? Encoding your unnatural amino acid of choice?

  9. dave w says:

    #7: Ah, that’s how you keep the Synthetic Orcs from proliferating uncontrollably…!

  10. molecular architect says:

    I’m a chemist and I think I must have missed something. So, can the bacteria actually use the new alphabet? Or is the breakthrough confined to the presence of the alternative nucleosides being tolerated?

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