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Watching mRNA Do Its Thing, In Living Cells

This is a nice chemical biology paper that hits on a hot topic of the day: the uptake and function of mRNA when administered to cells. You can always look for downstream effects to show that you achieved both those goals, but it would be very useful to get images of this process in real time. Here’s a heavily cited paper from 2013 that was able to do this for siRNA species, but the colloidal gold labeling technique used is not a straightforward one.

To that end, there have been many attempts at generating other fluorescent RNA and DNA species, but as usual, you always have to look out for side effects of the fluorescent labeling. These things take up space and alter physical properties, and there’s not much way around that, so you just have to see if you can live with the results. Cyanine dyes (attached by cycloaddition reactions) have been used on both RNA and DNA, but it’s already known that the fluorescence on the latter can vary according to the sequence surrounding them, and both the type and positioning of such labels can impede the function of mRNA in general (such as choking up the ribosomal machinery). In general, you can go hard and label RNA with things that will be easy to pick up with good signal/noise, at the cost of degrading its ability to function, or you can label it with a lighter hand at the cost of being able to see it in living cells.

This new paper references these and several other methods that have been tried, and proposes a new variety of fluorescent nucleobase. Those as the name implies, are things that look like the existing bases used to form RNA and DNA species, but the purine/pyrimidine part has been modified to be fluorescent. The same constraints apply – can you get one that’s bright enough and long-lasting enough that’s still acceptable to the (rather highly evolved) enzymes that handle such building blocks? This paper has data on a fluorescent cytosine analog incorporating a 1,3,-diaza-2-oxophenoxazine instead of cytidine, and it seems to make the cut. In fact, the paper shows data indicating that they can exchange all the C residues in a 1200-base stretch of mRNA and still see it translated in live human cells, which is pretty impressive. It also features a new solid-support method to synthesize nucleoside triphosphates, which sounds pretty useful (since that can be a painful process) which looks to be the subject of a forthcoming paper itself.

That big stretch of mRNA is, in fact, the transcript for everyone’s favorite fluorescent species, green fluorescent protein (GFP). The team did do some codon-optimizing to cut down on the chances of having two modified-C residues right next to each other, which is probably going to gum up the works no matter what, but they report that a nonoptimized transcript for calmodulin went through just fine, on the other hand. The RNA polymerase enzymes seem to take it up with nearly the same alacrity that they do regular cytosine. As the amount of incorporated fluorescent base goes up, the emission gets a bit red-shifted, and the quantum yields and fluorescent lifetimes drop a bit, all of which are to be expected – some of that is due to self-quenching by residues that are close to each other. They seem to have maxed out at about 75% incorporation; higher than that is self-defeating. But 25% incorporation is bright enough for cellular imaging, so you can stop things there and have less chance of downstream troubles.

As for the results, let me send you here so you can watch a time-lapse (from the supporting data, free to view/download). That’s part of a frame from the movie at right. Red mRNA comes into the cell in little round endosomal packets (as shown by separate orange fluorescent labeling of an early endosome biomarker), followed by the development of what is obviously correctly expressed and folded GFP over several hours. Watching red mRNA get processed into green protein is quite satisfying; I can only imagine how happy the authors must have been to see it work. This is, I believe, a first for the fluorescent-base technique in general. This sort of thing will surely be very useful for RNA applications in general, as you can (in theory) watch your proposed vaccine or drug sequence getting taken up by live cells, trafficked to the right locations in the cytosol, and subsequently degraded in real time. I get a lot of questions along the lines of “How do we know that these mRNAs are going to the right places?” Well, from what I can see, this could be the most straightforward way to answer them!

 

16 comments on “Watching mRNA Do Its Thing, In Living Cells”

  1. Simon says:

    Neat!
    Derek, how do I find individual blog posts? I want to re-read the odd structures in natural products and the smallest drig structure posts…

    1. Marcus Theory says:

      I’m curious too, as I’ve found Google to be better than anything on AAAS. Googling “smallest drug structure in the pipeline” yields this:

      https://blogs.sciencemag.org/pipeline/archives/2014/08/27/the_smallest_drug

      1. Hap says:

        I’ve generally used “site:blogs.sciencemag.org/pipeline [desired string, without brackets]” to find posts: for example, “site:blogs.sciencemag.org/pipeline nitric acid” (no quotes) returns this post and this post (and an ad from Aldrich).

      2. Ross Presser says:

        I use InoReader as my RSS reader, and have been subscribed to the feed for this blog for several years. I can scroll back in the inoreader page and see all the titles. I’ve tested and scrolled back to October 2019; it may go even further.

  2. Marcus Theory says:

    The effect is somewhat diminished for the red-green colorblind! But still cool.

  3. Simon says:

    Ha! That works great, didn’t think to use it for internal content.

  4. Eli Barnett says:

    This is the coolest thing you’ve posted in months. Not that your other content hasn’t been good, mind, but wow.

  5. Jonathan B says:

    Beautiful stuff, if a picture is worth a thousand words the best video is worth ten thousand. It triggers two different memories of my long-lost post-doc youth.

    I happened share a lab bay with Ari Helenius in late 1978 when he did the definitive experiments (the outcome of several months of exploring the conditions) showing that an enveloped viruse enters cells via a coated vesicle/endosomal route (https://rupress.org/jcb/article/84/2/404/21081/On-the-entry-of-semliki-forest-virus-into-BHK-21) and shared those images straight from the darkroom and the graphs drawn by hand on paper. Alynd then about eight or nine years later was a colleague of Brad Amos and John White when they built one of the earliest confocal microscopes (https://rupress.org/jcb/article/105/1/41/28932/An-evaluation-of-confocal-versus-conventional). That first prototype – which I was an early user of with my own samples – was built horizontal on a classic physics optical bench inside a tent made of black-out curtain (the third author, Mick Fordham, was the workshop technician who made the physics concept into a prototype reality). And some years later, in a totally different context I was a colleague of Alan Boyde who was probably the first to apply confocal optics to a biological investigation (https://science.sciencemag.org/content/230/4731/1270.long).

  6. Ken says:

    Next up, label the tRNAs with something that fluoresces yellow.

  7. Rich says:

    Has radioactive labelling been tried, e.g tritium or C14?

  8. Martin Bonner says:

    Does anyone have a link to the time-lapse? When I followed Derek’s link it just went to the publisher’s “give us money” page.

    1. Marko says:

      Try this ( may need to scroll down) :

      https://pubs.acs.org/doi/10.1021/jacs.1c00014

  9. A Nonny Mouse says:

    AZ vaccine making spike proteins with Cryo EM

    https://pubs.acs.org/doi/10.1021/acscentsci.1c00080#

  10. Nigel says:

    You’ve taken a dose of mRNA now have a dose of reality:
    https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(21)00468-2/fulltext
    If your vaccine is of any real practical use now it won’t take too long until you need a booster shot as the virus is still mutating very rapidly indeed. Uncertain effects of vaccines on transmissibility, unknown medium to long term immunity, current/future variant breakthrough infections, the travesty of global vaccine rollouts and idiotic policies such as vaccine bonuses, passports and green cards all point to there being another major flare up, probably next fall or winter. Throw in the real possibility of ADE and the consequences are potentially much worse than what we’ve seen to date.
    In any event (even in the unlikely scenario that vaccines actually work) you’ll be waiting a long while for herd immunity – Gavi say that nobody’s safe in the world until we are all safe. Most of the worlds population are only getting thrown tiny scraps from the first world table and South Africa refused to eat the AZ ones. Covid is a leveller of global health inequalities

  11. David Edwards says:

    And this is why I pay repeat visits to Derek’s blog.

    Even with my limited knowledge, I can see that this is an inspired piece of genius. And I think I know enough about the fun and games of the ribosome to understand that getting this to work was a non-trivial task to put it mildly.

    Once again, the famous XKCD maxim has proven true. 🙂

  12. Marcus Wilhelmsson says:

    Hi Derek,

    Thanks for your nice summary of our work! I can confirm that the first time we saw the green emission from the tagged protein in the nucleus was indeed one of the happiest moments in my research career so far. I hope and think this method will be useful for the community.

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