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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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October 9, 2008

More Glowing Cells: Chemistry Comes Through Again

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Posted by Derek

I’ve spoken before about the acetylene-azide “click” reaction popularized by Barry Sharpless and his co-workers out at Scripps. This has been taken up by the chemical biology field in a big way, and all sorts of ingenious applications are starting to emerge. The tight, specific ligation reaction that forms the triazole lets you modify biomolecules with minimal disruption (by hanging an azide or acetylene from them, both rather small groups), and tag them later on in a very controlled way.

Adrian Salic and co-worker Cindy Yao have just reported an impressive example. They’ve been looking at ethynyluracil (EU), the acetylene-modified form of the ubiquitous nucleotide found in RNA. If you feed this to living organisms, they take it up just as if it were uracil, and incorporate it into their RNA. (It’s uracil-like enough to not be taken up into DNA, as they’ve shown by control experiments). Exposing cells or tissue samples later on to a fluorescent-tagged azide (and the copper catalyst needed for quick triazole formation) lets you light up all the RNA in sight. You can choose the timing, the tissue, and your other parameters as you wish.

For example, Salic and Yao have exposed cultured cells to EU for varying lengths of time, and watched the time course of transcription. Even ten minutes of EU exposure is enough to see the nuclei start to light up, and a half hour clearly shows plenty of incoporation into RNA, with the cytoplasm starting to show as well. (The signal increases strongly over the first three hours or so, and then more slowly).

Isolating the RNA and looking at it with LC/MS lets you calibrate your fluorescence assays, and also check to see just how much EU is getting taken up. Overall, after a 24-hour exposure to the acetylene uracil, it looks like about one out of every 35 uracils in the total RNA content has been replaced with the label. There’s a bit less in the RNA species produced by the RNAPol1 enzyme as compared to the others, interestingly.

There are some other tricks you can run with this system. If you expose the cells for 3 hours, then wash the EU out of the medium and let them continue growing under normal conditions, you can watch the labeled RNA disappear as it turns over. As it turns out, most of it drops out of the nucleus during the first hour, while the cytoplasmic RNA seems to have a longer lifetime. If you expose the cells to EU for 24 hours, though, the nuclear fluorescence is still visible – barely – after 24 hours of washout, but the cytoplasmic RNA fluorescence never really goes away at all. There seems to be some stable RNA species out there – what exactly that is, we don’t know yet.

Finally, the authors tried this out on whole animals. Injecting a mouse with EU and harvesting organs five hours later gave some very interesting results. It worked wonderfully - whole tissue slices could be examined, as well as individual cells. Every organ they checked showed nuclear staining, at the very least. Some of the really transcriptionally active populations (hepatocytes, kidney tubules, and the crypt cells in the small intestine) were lit up very brightly indeed. Oddly, the most intense staining was in the spleen. What appear to be lymphocytes glowed powerfully, but other areas next to them were almost completely dark. The reason for this is unknown, and that’s very good news indeed.

That’s because when you come up with a new technique, you want it to tell you things that you didn’t know before. If it just does a better or more convenient job of telling you what you could have found out, that’s still OK, but it’s definitely second best. (And, naturally, if it just tells you what you already knew with the same amount of work, you’ve wasted your time). Clearly, this click-RNA method is telling us a lot of things that we don’t understand yet, and the variety of experiments that can be done with it has barely been sampled.

Closely related to this work is what’s going on in Carolyn Bertozzi’s lab in Berkeley. She’s gone a step further, getting rid of the copper catalyst for the triazole-forming reaction by ingeniously making strained, reactive acetylenes. They’ll spontaneously react if they see a nearby azide, but they’re still inert enough to be compatible with biomolecules. In a recent Science paper, her group reports feeded azide-substituted galactosamine to developing zebrafish. That amino sugar is well known to be used in the synthesis of glycoproteins, and the zebrafish embryos seemed to have no problem accepting the azide variant as a building block.

And they were able to run these same sorts of experiments – exposing the embryos to different concentrations of azido sugar, for different times, with different washout periods before labeling all gave a wealth of information about the development of mucin-type glycans. Using differently labled fluorescent acetylene reagents, they could stain different populations of glycan, and watch time courses and developmental trafficking – that’s the source of the spectacular images shown.

Bertozzi%2Cjpg.jpg

Losing the copper step is convenient, and also opens up possibilities for doing these reactions inside living cells (which is definitely something that Bertozzi’s lab is working on). The number of experiments you can imagine is staggering – here, I’ll do one off the top of my head to give you the idea. Azide-containing amino acids can be incorporated at specific places in bacterial proteins – here’s one where they replaced a phenylalanine in urate oxidase with para-azidophenylalanine. Can that be done in larger, more tractable cells? If so, why not try that on some proteins of interest – there are thousands of possibilities – then micro-inject one of the Bertozzi acetylene fluorescence reagents? Watching that diffuse through the cell, lighting things up as it found azide to react with would surely be of interest – wouldn’t it?

I’m writing about this the day after the green fluorescent protein Nobel for a reason, of course. This is a similar approach, but taken down to the size of individual molecules – you can’t label uracil with GFP and expect it to be taken up into RNA, that’s for sure. Advances in labeling and detection are one of the main things driving biology these days, and this will just accelerate things. (It’s also killing off a lot of traditional radioactive isotope labeling work, too, not that anyone’s going to miss it). For the foreseeable future, we’re going to be bombarded with more information than we know what to do with. It’ll be great – enjoy it!

Comments (7) + TrackBacks (0) | Category: Analytical Chemistry | Biological News


COMMENTS

1. SP on October 9, 2008 12:08 PM writes...

I'm surprised how routine the Schultz suppressor tRNA method is these days.

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2. eric.suh on October 9, 2008 2:33 PM writes...

Not only is click chemistry getting rid of radiolabeling, but it's also phasing out some antibody work (e.g. using antibodies against BrdU for measuring DNA synthesis). Using antibodies is a huge pain, and click chemistry is making a lot of it so much nicer.

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3. SciChick on October 9, 2008 4:40 PM writes...

Esp. for glycosylations, which are post-translational modifications that are also dynamic in nature - being able to see the changes in glycosylation pattern in REAL TIME in living cells, instead of having to do ex vivo labeling, that, is just too cool.

Bertozzi rocks! Though these days people only care about Huisgen cycloaddition (I refuse to call it click chemistry as there are many "click-like" reactions out there), the Staudinger ligation is still a very elegant reaction - it's actually much easier to make the phosphine probes than the octyne probes.

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4. bacon on October 9, 2008 5:41 PM writes...

SciChick: The Staudinger ligation is too slow. Which is why Bertozzi is working on the cyclooctyne cycloadditions, which are quite impressive.

And the new reaction from Joseph Fox at Delaware is also very impressive. Much faster than even cyclooctyne + azide.

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5. Liquidcarbon on October 13, 2008 6:53 AM writes...

Recent cyclooctyne syntheses are much better compared to the first one. Tetrazine chemistry is insanely fast, thousands time faster than cyclooctyne+azide (up to 2000 L/mol*sec)! The reactions are complete in seconds at low micromolar concentrations! We'll see where will it go.

BTW, the first author on the second generation paper (JACS 2008 130 11486, 10.1021/ja803086r) was an undergrad.

Permalink to Comment

6. SciChick on October 13, 2008 5:28 PM writes...

One potential problem with the tetrazine system is that it's not small, and most likely not tolerable by enzymatic machinery for post-translational modifications (glycosylations, for example). Which is why the small azide (or acetylene) is such a wonderful chemical handle. For in vitro labeling, sure, the tetrazine system blows everything else out of the water; but for real time, live-cell imaging, it's most likely not going to work (hence why the paper was in JACS, instead of in Science or PNAS, IMHO).

Permalink to Comment

7. Don Lewis on October 28, 2008 3:30 PM writes...

A flood of click chemistry patent applications could slow the commercialization of these technologies. Scripps has an issued patent for Sharpless' work; UC has a published patent application for Bertozzi's work; it would be a good guess that Harvard has now filed on Salic's work; and there are at least 82 other published click chemistry patent app's pending. Its getting rather crowded.

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