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March 20, 2013
Using DNA to Make Your Polymers. No Enzymes Needed.
Here's an ingenious use for DNA that never would have occurred to me. David Liu and co-workers have been using DNA-templated reactions for some time, though, so it's the sort of thing that would have occurred to them: using the information of a DNA sequence to make other kinds of polymers entirely.
The schematic above gives you the idea. Each substrate has a peptide nucleic acid (PNA) pentamer, which recognizes a particular DNA codon, and some sort of small-molecule monomer piece for the eventual polymer, with cleavable linkers holding these two domains together. The idea is that when these things line up on the DNA, their reactive ends will be placed in proximity to each other, setting up the bond formation in the order that you want.
Even so, they found that if you use building blocks whose ends can react with each other intramolecularly (A----B), they tend to do that as a side reaction and mess things up. So the most successful runs had an A----A type compound on one codon, with a B----B one on the next, and so on. So what chemical reactions were suitable? Amide formation didn't get very far, and reductive amination failed completely. Hydrazone and oxime formation actually worked, though, although you can tell that Liu et al. weren't too exciting about pursuing that avenue much further. But the good ol' copper-catalyzed acetylene/azide "click" reaction came through, and appears to have been the most reliable of all.
That platform was used to work out some of the other features of the system. Chain length on the individual pieces turned out not to be too big a factor (Whitesides may have been right again on this one). A nice mix-and-match experiment with various azides and acetylenes on different PNA codon recognition sequences showed that the DNA was indeed templating things the in the way that you would expect from molecular recognition. Pushing the system by putting rather densely functionalized spacers (beta-peptide sequences) in the A----A and B----B motifs also worked well, as did pushing things to make 4-, 8-, and even 16-mers. By the end, they'd produced completely defined triazole-linked beta-peptide polymers of 90 residues, with a molecular weight of 26 kD, which pushes things into the realm of biomolecular sizes.
You can, as it turns out, take a sample of such a beast (with the DNA still attached) and subject it to PCR, amplifying your template again. That's important, because it's the sort of thing you could imagine doing with a library of these things, using some sort of in vitro selection criterion for activity, and then identifying the sequence of the best one by using the attached DNA as a bar-code readout. This begins to give access to a number of large and potentially bioactive molecules that otherwise would be basically impossible to synthesize in any defined form. Getting started is not trivial, but once you get things going, it looks like you could generate a lot of unusual stuff. I look forward to seeing people take up the challenge!
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