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DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

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« Scientific Snobbery | Main | Off Topic: Happy New Year, In March »

March 20, 2013

Using DNA to Make Your Polymers. No Enzymes Needed.

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Posted by Derek

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.
Liu%20scheme.png
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!

Comments (6) + TrackBacks (0) | Category: Chemical Biology


COMMENTS

1. Boghog on March 20, 2013 12:31 PM writes...

ribosome-less translation, cool

biology is turning into chemistry

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2. Anon on March 20, 2013 2:56 PM writes...

Amide bond formation and reductive amination work fine when the reactive molecule is attached to a hybridizing DNA oligomer. Guess PNA hybridizes much more slowly and with less specificity. I didn't read the paper, so this might be stupid question, but why are they using PNA?? DNA is about as stable as you can get and its specificity is higher? Derek, I like your recent emphasis on the importance of greater diversity of screening collections and using novel means to achieve this.

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3. Curt F. on March 20, 2013 5:56 PM writes...

This looks super cool - thanks Derek for posting it. On the the note of insanely complex polymers, I also saw this work profiled in C&EN and was similarly amazed: http://cen.acs.org/articles/91/i11/Bold-Territory-Polymers.html (although the latter example doesn't use templates and is not selectable).

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4. Helical_Investor on March 20, 2013 6:14 PM writes...

Anon,

Guess PNA hybridizes much more slowly and with less specificity.

Exactly the opposite. I worked with PNA for years. Been out of touch with it though and could not tell you why they chose it.

Zz

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5. JB on March 20, 2013 8:04 PM writes...

Heh, the foundation for this was the first project I started on when I was a first year grad student and he was a first year professor. I ended up working on something more biological, less synthetic, but that gives you the context that they've been keeping this going for almost 15 years now. Two previous papers showed non-enzymatic polymerization of PNA on a DNA backbone and then generation of a library and selection of a spiked poscon-containing sequence.
My guess is you can make PNA at much larger scale cheaper- DNA building blocks are chiral and thus not cheap when you're talking about making enough to evolve physical properties as you would want to do with polymers (as opposed to enzymatic activity that you would normally evolve with biomolecules.) They might address this in the paper, haven't downloaded the full text yet. And yes, PNA (and LNA) are more specific/higher Tm- one argument for PNA is no negative charge to repel phosphate groups on the complementary strand.

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6. Adam on March 21, 2013 2:22 PM writes...

The reason of using PNA could be that they have to make the entire macrocycle in one batch, and it'll be more convenient and more efficient to use PNA 'cause they are coupled just like the other building blocks via peptide chemistry.

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