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Genes Can Be Synthetic Chemicals, You Know

Here’s one of those papers where you go “I’m really surprised that that even works”. But I shouldn’t be, I suppose, based on what’s led up to it. I last wrote about the work coming out of the University of Southampton on “clicked DNA” three years ago, but they’ve been busy. This latest paper shows that you can take ten different oligonucleotides, functionalized with azides and alkynes on their ends, and assemble them via triazole formation into a 350-base pair gene sequence (in this case, one for a green fluorescent protein). It has, then eight triazole linkages along its span.

When you do that, you get a surprisingly “life-like” DNA sequence. It’s got a bit lower melting point, but the secondary structure appears quite similar to the native stuff. This synthetic gene is recognized by the relevant enzymes in vitro, although PCR is slower, and slows down more with every triazole linkage. But the group didn’t stop there: when this clicked-DNA sequence is put into a plasmid for E. coli, the bacteria take it up and express the protein, which comes out the other end of the transcriptional machinery  justas it’s supposed to. Comparing this to transfection with a conventional plasmid, with the gene assembled through T4 DNA ligase, showed that the error rate is the same or better with the triazole-linked sequence. The cellular machinery doesn’t care. They did look into the possibility that the gene was being assembled through nucleotide excision repair as a way of dealing with the triazole linkages, but a bacterial strain lacking this pathway also expressed the synthetic gene, with no big differences between it and the ligase-assembled version.

The next part of the paper shows that you can use the power of a fully synthetic gene to incorporate epigenetic markers like 5-methylcytosine wherever you want. This is much more of a pain with the existing enzymatic systems, as slick as they are with unmodified DNA. The authors make the case that synthetic assembly of genes (which doesn’t necessarily have to be with click-trizoles, one would think) allows you entry into all sorts of modifications that the natural enzyme tools will balk at, and I think that they’re right. This should be of great interest to the epigenetics field, and to anyone looking to work with chemically modified DNA in general. The transcriptional machinery will put up with a lot more than we might have thought, and we should take advantage of its cooperation.

And conceptually, this continues to make the case that molecular biology is slowly turning into a branch of chemistry. I don’t think that enough chemists are aware of that, and I’m not sure that all molecular biologists are thrilled with that characterization, either, but here we have it. . .



Note: All opinions, choices of topic, etc. are strictly my own – I don’t in any way speak for my employer

12 comments on “Genes Can Be Synthetic Chemicals, You Know”

  1. Chrispy says:

    The paper is more interesting for the demonstration of the permissiveness of the transcription/translation machinery than as a useful DNA synthesis method. Click chemistry has come (to me) to look like an ugly kludge in the synthetic chemistry world. It is is used in all kinds of amateurish drug discovery efforts, from antibody-drug conjugates to fragment-based approaches, and it almost seems impolite to ask: “So what effect are the triazoles having?” In the case in the paper, a practical approach would have been to employ nick repair to the same set of primers they hook together with triazoles.
    A more interesting use of this would be to “shuffle” together bits of DNA that would not require any overlap or homology to combine — like a single-stranded blunt ligation.
    BTW, this article is on SciHub, for those of your readers who do not feel that shelling out $150 to Nature Chemistry represents a good value.

    1. tlp says:

      Funny thing is that authors didn’t show unambiguously that translation (click-DNA -> RNA synthesis) is permitted at all, they only showed that DNA can be replicated from click-DNA. They did reference one paper in the introduction but no experiment of theirs confirms that cells synthesize RNA based on their tetra-triazol-linked-DNA.

    2. zero says:

      > The paper is more interesting for the demonstration of the permissiveness of the transcription/translation machinery than as a useful DNA synthesis method.

      As a layman, this. I had always assumed that transcription and related enzymes would be exquisitely tuned to natural DNA. Reinforcing that assumption, alternative nucleotides have required synthetic enzymes for this purpose.

      Sounds like there are parts of these molecules which are sensitive to small changes and other parts that are quite tolerant. The tolerant bits seem like worthwhile targets of study; who knows what that might reveal about the evolution of genetic material, not to mention the epigenetic factors already mentioned.

    3. Bla bla says:

      Click chemistry has been an absolute godsend for me in at least one area of my work. As a stem cell biologist, I spend a lot of time looking at cell proliferation. Used to do this by bromodeoxyuridine incorporation (yes yes, DNA repair too, I know). Which required acid or heat treatment to denature the DNA enough for an antibody to come in to detect it. Which more often then not screws up all sorts of other stuff we want to look at.

      Now? We hit the cells/animals with ethynyl-deoxyuridine, slap on some copper, vit C and an azide fluorophore, and it’s all done and dusted in 30 min. I won’t hear a bad word about triazoles at least for this application!

  2. Kyle MacDonald says:

    Newbie question: how likely is it that the epigenetic markers will be handled as cleanly as the sequence of base pairs? How much of the space of possible epigenetic wizardry did these authors explore, and are there any markers that you expect might not play well with this technique?

  3. Barry says:

    Of course, “molecular biology” has always been a field of chemistry. That’s what the “molecular” originally signified. It’s no accident that so many of the seminal contributions were from physicists

  4. tlp says:

    Nitpicking: aren’t their qPCR and PCR results quantitatively inconsistent? How can one get delta-Ct of 14.4 (i.e. ~5 orders of magnitude in amplification speed, Fig. 3f(iv)) and equal intensities of PCR bands (Fig. S3)?
    And yes, epigenetic speculation seems irrelevant, because when DNA is replicated the epigenetic marks are erased, aren’t they? Unless anyone wants to study epigenetics in vitro with fully synthetic DNA.

    1. tlp says:

      ouch, my bad, at 30 cycles both DNA and click-DNA curves are saturated anyway

  5. milkshaken says:

    I am willing to bet that the paper results are not reproducible

    They use regular copper-catalyzed click chemistry. The trouble is that Cu coordinates to nucleic acids and in presence of air and ascorbate generates reactive oxygen species that degrade nucleic acids

  6. Arda says:

    From a biochemist’s point of view, this could be really useful for bringing down the cost of gene synthesis. It should be fairly straightforward to synthesise a small oligonucleotide by solid phase synthesis and then click further oligos onto it to generate a large gene. This can then be amplified by PCR to conveniently remove the triazoles

  7. Ali Tavassoli says:

    Thank you to everyone above (especially Derek) for their interest in our work. I thought I should answer some of the questions that have been raised in the comments.

    #3 – Our collaborators (Prof. Tom Brown and his group) have shown that T7 RNA polymerase reads through the linker (10.1039/c1cc14316f) and we have shown transcription through the linker in human cells (10.1002/anie.201308691). Accurate translation has been confirmed by mass spectrometry (first paper) and reverse transcription and sequencing (in the second paper). In this paper the experiments are in E. coli, so the click-linked gene will be rapidly replicated to canonical DNA (50-100 copies) and it will be impossible to distinguish between mRNA from click-linked DNA and that from replicated, canonical DNA. It is not the same case in human cells where there will be no DNA replication, so it will be easy to demonstrate transcription (stay tuned for this data).

    #5 – you raise a good point. The triazole linkers may impact reading of the encoded epigenetic signal. We need to look into this, and if this is the case, we will consider this when designing where to ‘click-ligate’ oligos in relation to the position of any epigenetic markers.

    #10 – You are correct, copper can bind to DNA, which is why we use such high salt concentration during our ‘click-ligation’ step. We have always found this to be a bigger issue in terms of sequestering the catalyst rather than causing DNA damage. If one is particularly worried about oxidation, the reaction can always be carried out in an inert atmosphere. Prof Brown’s lab have recently published (10.1021/jacs.6b11530) next gen sequencing data on PCR products of replication through this linker if you would like additional information on this.
    I should also point out that click-ligation of oligonucleotides has been reproduced by Prof Brown, us and several other laboratories around the world.

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