4 ms·

People are missing the point. It's not the click that matters. It's the bio-orthogonal part. This lets me grow cells with one of the click substrates, say, a m

People are missing the point. It's not the click that matters. It's the bio-orthogonal part. This lets me grow cells with one of the click substrates, say, a methionine substitute that incorporates into every protein, purify out my protein, and then easily do chemistry on that. That's near invaluable. I can grow a virus and put a different click substrate in each of the DNA, protein, and lipids. Then I can infect cells, and mid-infection, separately label each macromolecule a different color. Then, I can figure out what parts of the virus go into the cell, which sheds light on the mechanism of entry. Having it bio-orthogonal means the biology is preserved and nothing is messed up until the moment of observation.

panabee 4y ago
Thanks for the explanation. This seems amazing, but could you kindly clarify how adding glycans to a cell is guaranteed to not disturb biological function?
throwawaymaths 4y ago
What "different click substrates" are you talking about? As far as I remember There's only one class that's been developed (alkyne azide cycloaddition) -- sharpless really wants norbornene to take off but no one has developed that afaict - and of these only one that can be done in vivo (cyclooctyne + azide); the others require copper catalyst which is not awesome for living cells. > ... methionine substitute that incorporates into every protein, purify out This is crazy labor intensive, I feel like if you are at this point in considering using this to understand a biological you have really burned through a lot of simpler, more established, and easy to calibrate techniques.
- sdbrown 4y ago
  Tetrazine/DBCO is also pretty popular IIRC.
  - throwawaymaths 4y ago
    Fair. This holds a place in my mind as "basically the same as alkyne - azide" since Tetrazzine is "more or less azide" and dbco is "more or less cyclooctyne". More to the point of the thread, I don't think the Tetrazzine/dbco is orthogonal to alkyne-azide -- they will cross-react -- am I wrong about that?
    - sdbrown 4y ago
      Apologies -- tetrazine reacts principally with trans-cyclooctenes. So azide/DBCO would be the orthogonal group. To the best of my knowledge, those two pairs do not cross-react.
- COGlory 4y ago
  >What "different click substrates" are you talking about? I can click on a strep tag, or a his tag, or a fluorophore of any different color. See: https://clickchemistrytools.com/ https://clickchemistrytools.com/ Yes, they are all the same chemical addition, but I can label them orthogonally. I can incorporate an azo-amino acid, and click a green alkyne. I can incorporate an alkyne nucleotide, and click a blue azide, etc. >This is crazy labor intensive, I feel like if you are at this point in considering using this to understand a biological you have really burned through a lot of simpler, more established, and easy to calibrate techniques. I don't think you are understanding what I'm saying here. What are you saying is labor intensive, purifying protein? That's an incredibly common technique. Yes it's labor intensive, but it's the only way to do huge amounts of experiments. If you're saying click chemistry is labor intensive, well, you're wrong, it's a couple hour experiment, most of which is waiting for incubations.

panabee 4y ago

Thanks for the explanation. This seems amazing, but could you kindly clarify how adding glycans to a cell is guaranteed to not disturb biological function?

throwawaymaths 4y ago

What "different click substrates" are you talking about? As far as I remember There's only one class that's been developed (alkyne azide cycloaddition) -- sharpless really wants norbornene to take off but no one has developed that afaict - and of these only one that can be done in vivo (cyclooctyne + azide); the others require copper catalyst which is not awesome for living cells. > ... methionine substitute that incorporates into every protein, purify out This is crazy labor intensive, I feel like if you are at this point in considering using this to understand a biological you have really burned through a lot of simpler, more established, and easy to calibrate techniques.

sdbrown 4y ago
Tetrazine/DBCO is also pretty popular IIRC.
- throwawaymaths 4y ago
  Fair. This holds a place in my mind as "basically the same as alkyne - azide" since Tetrazzine is "more or less azide" and dbco is "more or less cyclooctyne". More to the point of the thread, I don't think the Tetrazzine/dbco is orthogonal to alkyne-azide -- they will cross-react -- am I wrong about that?
  - sdbrown 4y ago
    Apologies -- tetrazine reacts principally with trans-cyclooctenes. So azide/DBCO would be the orthogonal group. To the best of my knowledge, those two pairs do not cross-react.
COGlory 4y ago
>What "different click substrates" are you talking about? I can click on a strep tag, or a his tag, or a fluorophore of any different color. See: https://clickchemistrytools.com/ https://clickchemistrytools.com/ Yes, they are all the same chemical addition, but I can label them orthogonally. I can incorporate an azo-amino acid, and click a green alkyne. I can incorporate an alkyne nucleotide, and click a blue azide, etc. >This is crazy labor intensive, I feel like if you are at this point in considering using this to understand a biological you have really burned through a lot of simpler, more established, and easy to calibrate techniques. I don't think you are understanding what I'm saying here. What are you saying is labor intensive, purifying protein? That's an incredibly common technique. Yes it's labor intensive, but it's the only way to do huge amounts of experiments. If you're saying click chemistry is labor intensive, well, you're wrong, it's a couple hour experiment, most of which is waiting for incubations.