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