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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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January 23, 2014

Clicked-DNA Works In Human Cells

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Posted by Derek

I made reference to the (surprising) ability of artificially-linked "click" DNA analogs (an earlier example here) to show activity in cells when Ali Tavassoli (of U. Southampton) spoke about his lab's work back at the Challenges in Chemical Biology conference last summer.

Here's their 2012 paper on the subject, and now they have another one out in Angewandte Chemie, which extends the work to human cells. It sort of boggles me mind to think that these things are actually transcriptionally active, but they're lighting up human cells with the fluorescent dye mCherry, and doing (from what I can see) all the appropriate control experiments. (It's not being reworked by repair enzymes along the way, for example). Here's the wrap-up:

The practical limit on the length of error-free oligonu- cleotide synthesis has necessitated the use of enzymes for the assembly of polynucleotide chains into genes. However, these approaches have been constrained by the assumption that the phosphodiester backbone that links oligonucleotides is critical for the biocompatibility and cellular function of the resulting DNA. As demonstrated in this work, this is not the case. Our results strongly suggest that RNA polymerase II, the enzyme responsible for all mRNA synthesis in eukaryotes, correctly transcribes the genetic information contained on a click-linked strand of DNA. . .Our results indicate that a phosphodiester linker is not essential for joining oligonucleotides for gene synthesis and open up the possibility of replacing enzymatic ligation with highly efficient chemical reactions. This approach would not necessarily be limited to the linker reported here, and alternative chemical reactions and the resulting linkers may also be suitable for this purpose.

I look forward to seeing what use chemical biology will make of this sort of thing. Now, can you make functional mRNAs out of this as well?

Comments (17) + TrackBacks (0) | Category: Chemical Biology


COMMENTS

1. Puff the Mutant Dragon on January 23, 2014 11:54 AM writes...

This is unbelievably cool.

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2. Anonymous on January 23, 2014 12:31 PM writes...

It's a really cool experiment, but it seems disingenuous to imply that this obviates the need for a phosphodiester backbone - they tested DNA blocks with a *single* click-linked lesion, and seemed to observe a marked decrease in mCherry expression off of those constructs... As a chemical biologist, I'd be wary of using anything with more than a single lesion, and it seems to me that the really interesting result would be if you could replace many or even most of the phosphodiester linkages in DNA with some sort of synthetic analog. Still, it's absolutely amazing that it works, and could one day yield some very interesting sorts of experiments.

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3. Anonymous on January 23, 2014 12:54 PM writes...

It's very cool.
To the person above - they are not saying this negates the need for a phosphodiester backbone. They are saying that instead of using ligases, or PCA to join oligonucleotides for gene assembly click chemistry can be used: "phosphodiester linker is not essential for joining oligonucleotides for gene synthesis". You would only ever have one of these linkers every 100 bases or so. i.e. a purely synthetic method for gene synthesis. This would be huge.

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4. Anonymous on January 23, 2014 1:19 PM writes...

#3: I get that, but don't see the advantage. Ligases are incredibly easy to use, and functionally very efficient. This would be useful only for those cases where you can't use ligases, which to me seems to be a rather limited subset of experiments. I mean, don't get me wrong, it's a very cool observation and all, but I don't really see a need for it in that role - it strikes me as serving a very niche role in the canon of DNA chemistry.

Also, on closer reading of the paper, I do have some methodological questions about it... It seems that their readouts for activity are really readouts for transfection efficiency. This speaks very little to the actual biological tolerance of these sorts of materials, only that their plasmids correctly behave like plasmids... I'd love to have seen either an RT-qPCR or Flow cytometric analysis of their transfected cell populations, to determine if there's any variation in the actual transcription of the gene between their positive control and click ligated samples. Since they never quantitate beyond 'mCherry+' and 'mCherry-', it's impossible to say what the relative transcription efficiency of these plasmids is (from personal experience, microscopy can pick up surprisingly low levels of mCherry expression), and the use of microscopy rather than a more quantitative measure as their readout introduces a lot of potential variability into the experiment.

Overall, this strikes me as a promising start for this sort of research, but the paper seems incredibly premature - and needs a *ton* of followup work in order to suss out fully what's going on.

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5. MB on January 23, 2014 2:28 PM writes...

Yet another application of click chemistry. Makes you wonder why Sharpless hasn't won the Nobel for it yet. Cool paper.

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6. Hank Moody on January 23, 2014 4:54 PM writes...

#4- they have microinjected, not transfected. And ligases are ok if you are cloning, but never used for gene assembly as they suck for anything more than 3 fragments. I agree with you that more needs to be done to show level of fluorescence, but give them a break - its a communication not a full paper. Its pretty cool that it works.

#5- Morten Medal deserves as much credit as Sharpless. But he is a pretty humble guy and doesnt get the general recognition that he dserves.

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7. Anonymous on January 23, 2014 6:06 PM writes...

More interesting (to me at least) than transcription would have been to test for recognition by DNA polymerases, which, if it works, would drastically reduce costs for synthesis of large DNA molecules. I am already looking forward to a world in which we won't have to painstakingly clone genes and instead just sequence everything and then order a synthetic plasmid of whatever we want.

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8. Rhenium on January 23, 2014 6:25 PM writes...

Pop Quiz Time:

As soon as you get to the end of this sentence, name another "click chemistry" reaction other than the copper catalyzed Huisgen cycloaddition.

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9. leftscienceawhileago on January 23, 2014 6:39 PM writes...

Agreed, CuAAC is generally synonymous with "click" (people seem to get offended by this)

The only really interesting click stuff is bioorthogonal stuff...tetrazine click is pretty neat, and some super fast hetero-diels alder as well (not regio specific). There really isn't anything out there that compares to CuAAC though...but I don't think it is impossible that we might find something some day...maybe a neat asymmetric organo catalysis click...

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10. MB on January 23, 2014 8:40 PM writes...

Tetrazine and inverse diels alder click reactions are neat but the synthesis for some of the reagents is an absolute beast. Ring strained click reactions are also neat but I doubt the feasibility due to the pharmacological liabilities of the reagents. In some cases, the staudinger ligation has shown to be superior than ring strained reactions in vivo, even though ring strained reactions are supposed to be kinetically superior. CuAAC can be modified with special ligands for Cu (I) to make it biocompatible.

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11. AnotherChemist on January 23, 2014 10:54 PM writes...

Well, at least this is more believable than arsenic-DNA life forms on Earth--anyone remember that imbroglio?

The Staudinger Ligation would be more useful if only phosphines were oxidation-resistant and the reaction were faster. While copper-free cycloadditions are useful, the cyclooctyne/octene syntheses are usually clunky. (Personally not a fan of running silver-treated silica columns.)

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12. Patrick on January 26, 2014 11:03 AM writes...

Sadly, I won't have access to the paper through our paywall for a while.

Did they demonstrate transient expression as well as stable integration? Or did they provide any other evidence suggesting that the synthetic construct was able to integrate/be replicated within the nucleus?

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13. I hate protein on January 26, 2014 4:19 PM writes...

#8- Strained cyclooctynes? lol

#9- Calling copper "bioorthogonal" is also pretty offensive to some people

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14. Christoph on January 27, 2014 4:23 PM writes...

#10 - Synthesis of strained cyclooctenes (TCO, s-TCO) as well as cyclopropene tags is pretty straight forward... Tetrazines might be a bit more tricky, but Zn/Ni catalysis usually gives decent yields

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15. Tom Brown on January 29, 2014 4:14 PM writes...

Just a few comments on previous posts, we have previously published papers to show that the triazole DNA linkage decribed in the Angew. Chem. paper can be read through by DNA polymerases (including thermostable ones), in linear, PCR and RCA modes, and by T7 RNA polymerase. We have also ligated and crosslinked DNA strands using Diels-Alder chemistry and copper free SPAAC chemistry (cyclooctyne + azide). This includes the assembly of various DNA/gold nanoconstructs. We have used the CuAAC reaction to assemble RNA constructs from smaller RNA strands (hairpin ribozyme), so the most recent publication is the latest in a series of papers going back to 2007.

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16. Phillip on February 11, 2014 10:57 AM writes...

@12 Patrick - the abstract landing page seems to suggest that this paper is free to access - don't know if that's been added since it went up, or if it's only for a limited time, but it does seem to be free now.

I know the RSC does this for any RSC papers that are highlighted in Chemistry World - they are made free to read for 6 weeks.

Permalink to Comment

17. prabu on March 28, 2014 5:24 AM writes...

As a substance scientist, I'd be careful about utilizing anything with more than a solitary injury, and it appears to me that the truly fascinating effect might be whether you could trade numerous or even the majority of the phosphorescent linkages in DNA with a manufactured simple.

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