But RNA also needs a boot on the right leg. And that boot has to be stable enough to stay on when you take the left one off so you don’t add to the wrong foot or have that foot kick off the base and breaking the chain. You don’t have to worry about this with DNA because its 3’ leg’s just a hydrogen (H) which isn’t reactive, but that oxygen is, so it can attack the phosphate. Different protective “boots” can be used to keep the 2’ OH protected (and the rest of the RNA protected from it!). If you’ve ever been in one of those walking cast boots you’ll be able to appreciate that bulky things can get in the way and slow things down. So bulky groups like TBS (ter-butyldimethylsilyl) (the traditional boot) can make the RNA synthesis takes longer. So there are improved (but more expensive) modified boots like 2-O-triisopropylsilyloxymethyl (TOM) (faster synthesis because spacer means it gets in the way less and 2′-bis(2-Acetoxyethoxy)methyl (ACE) protecting groups which are so stable you can order the RNA still protected if you want to make modifications to it yourself. On the other side of the table are 2′-thiomorpholine-4-carbothioate (TC) groups, which you can remove at the same time as you remove the base protectors. The “solid-phase” part is that before you start joining together bases, the start end is physically tied down to a solid base → helps the reacting molecules find each other (like “hug-a-tree” if you’re lost) and lets you wash off unreacted stuff without losing your product. it also helps ensure that nothing’s added “before it” in the chain (your end remains the end) then a full “synthetic cycle” (consisting of 4 steps) is performed for each base addition (more details in pics): DETRITYLATION - unhide (deprotect) the 5’ “arm” of the tied-down letter - remove the 5’ glove to reveal the OH COUPLING - add a new nucleoside (as a phosphoramidite monomer) to the unhidden 5’ OH OXIDATION → stabilized newly formed bond - it’s initially added as a phosphIte trimester, but that’s unstable → oxidization converts it to stable phosphAte triester at this point, for DNA it’s just like a “natural” DNA backbone except for 1 important difference → to prevent unwanted interactions, the phosphate group is still hidden (protected) by a Β-cyanoethyl group. For RNA the backbone still has that protective group on the 2’ OH that you want to stay there! (for now) CAPPING → hide uncoupled chains to prevent typos → coupling isn’t completely efficient (even when you dump in a huge excess of incoming nucleosides, not all the chains will get an added link) → if nothing got added to the tied-down nucleoside this cycle, you want to prevent something from being added the next cycle or else you’ll end up with a “skipped letter” typo. To avoid this you have to remove it from the reactant pool, but it’s physically tied down, so instead of physically removing it, you chemically hide it by blocking the 5’-OH (with an acetyl group for DNA or a tert-butylphenoxyacetic anhydride for RNA). Don’t worry, the properly coupled ones won’t be affected because their 5’-OH is already in use (bound to the new nucleoside) You keep doing this cycle (washing out “excess” nucleosides in between) until you’ve added each letter in your desired sequence. Then at the end you have to perform CLEAVAGE to untie the 3’ end from the solid support through ester hydrolysis of the linker → produces oligonucleotide w/terminal free 3’-OH Finally, you have a DEPROTECTION step to remove the “hiders” - remove the cyanooethyl groups (protecting the phosphates) by adding concentrated ammonia to make them want to “fall off” and deprotect the bases Now you need to purify your reaction products to remove “shortmers” - oligos that are the wrong length because you had to terminate them early because they failed to couple in a cycle. Purification methods include HPLC (High Performance Liquid Chromatography, which is a column-based method kinda like we use for protein purification, but with different columns and solvents and stuff) and PAGE purification, which useless PolyAcrylamide Gel Electrophoresis - the oligos get run through a gel which separates the fragments by size and then the correct-size bands can be removed and further cleaned. Such chemical synthesis works great for making really short pieces of DNA (like primers for PCR) but since more and more “drop out” each round because you have to terminate them because they failed to couple the yield (how much product you’ll get) drops off the longer you go (and there’s more chance of errors). So, the upper limit’s about 200 nucleotides. But more commonly you’re ordering things that are only 20-ish nucleotides long, frequently PCR primers. bit.ly/pcrprimer Another way to get custom RNAs made is with in-vitro transcription, which uses a phage (bacteria-infecting virus) RNA polymerase to make template-based copies similarly to how it happens in our cells, but in this case you’re doing it in a test tube. It can be used for making longer RNAs, but your transcript has to start with letter(s) the polymerase likes (so, G if you’re using T7 RNAP). bit.ly/t7rnap LOTS more on DNA & RNA bit.ly/nucleicacidstructure more on oligonucleotides: blog: bit.ly/oligomeaning ; TH-cam: th-cam.com/video/W0WbdR1aj20/w-d-xo.html more on ASOs: more on antisense oligonucleotides (ASOs): bit.ly/ASO_biochemistry & th-cam.com/video/KJGsTNmU_2M/w-d-xo.html resources: atdbio has a really nice guide: atdbio.com/nucleic-acids-book/Solid-phase-oligonucleotide-synthesis and here’s one from Sigma on DNA Oligonucleotide Synthesis: www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/pcr/dna-oligonucleotide-synthesis
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com
But RNA also needs a boot on the right leg. And that boot has to be stable enough to stay on when you take the left one off so you don’t add to the wrong foot or have that foot kick off the base and breaking the chain. You don’t have to worry about this with DNA because its 3’ leg’s just a hydrogen (H) which isn’t reactive, but that oxygen is, so it can attack the phosphate.
Different protective “boots” can be used to keep the 2’ OH protected (and the rest of the RNA protected from it!). If you’ve ever been in one of those walking cast boots you’ll be able to appreciate that bulky things can get in the way and slow things down. So bulky groups like TBS (ter-butyldimethylsilyl) (the traditional boot) can make the RNA synthesis takes longer. So there are improved (but more expensive) modified boots like 2-O-triisopropylsilyloxymethyl (TOM) (faster synthesis because spacer means it gets in the way less and 2′-bis(2-Acetoxyethoxy)methyl (ACE) protecting groups which are so stable you can order the RNA still protected if you want to make modifications to it yourself. On the other side of the table are 2′-thiomorpholine-4-carbothioate (TC) groups, which you can remove at the same time as you remove the base protectors.
The “solid-phase” part is that before you start joining together bases, the start end is physically tied down to a solid base → helps the reacting molecules find each other (like “hug-a-tree” if you’re lost) and lets you wash off unreacted stuff without losing your product. it also helps ensure that nothing’s added “before it” in the chain (your end remains the end)
then a full “synthetic cycle” (consisting of 4 steps) is performed for each base addition (more details in pics):
DETRITYLATION - unhide (deprotect) the 5’ “arm” of the tied-down letter - remove the 5’ glove to reveal the OH
COUPLING - add a new nucleoside (as a phosphoramidite monomer) to the unhidden 5’ OH
OXIDATION → stabilized newly formed bond - it’s initially added as a phosphIte trimester, but that’s unstable → oxidization converts it to stable phosphAte triester
at this point, for DNA it’s just like a “natural” DNA backbone except for 1 important difference → to prevent unwanted interactions, the phosphate group is still hidden (protected) by a Β-cyanoethyl group. For RNA the backbone still has that protective group on the 2’ OH that you want to stay there! (for now)
CAPPING → hide uncoupled chains to prevent typos → coupling isn’t completely efficient (even when you dump in a huge excess of incoming nucleosides, not all the chains will get an added link) → if nothing got added to the tied-down nucleoside this cycle, you want to prevent something from being added the next cycle or else you’ll end up with a “skipped letter” typo. To avoid this you have to remove it from the reactant pool, but it’s physically tied down, so instead of physically removing it, you chemically hide it by blocking the 5’-OH (with an acetyl group for DNA or a tert-butylphenoxyacetic anhydride for RNA). Don’t worry, the properly coupled ones won’t be affected because their 5’-OH is already in use (bound to the new nucleoside)
You keep doing this cycle (washing out “excess” nucleosides in between) until you’ve added each letter in your desired sequence.
Then at the end you have to perform CLEAVAGE to untie the 3’ end from the solid support through ester hydrolysis of the linker → produces oligonucleotide w/terminal free 3’-OH
Finally, you have a DEPROTECTION step to remove the “hiders” - remove the cyanooethyl groups (protecting the phosphates) by adding concentrated ammonia to make them want to “fall off” and deprotect the bases
Now you need to purify your reaction products to remove “shortmers” - oligos that are the wrong length because you had to terminate them early because they failed to couple in a cycle. Purification methods include HPLC (High Performance Liquid Chromatography, which is a column-based method kinda like we use for protein purification, but with different columns and solvents and stuff) and PAGE purification, which useless PolyAcrylamide Gel Electrophoresis - the oligos get run through a gel which separates the fragments by size and then the correct-size bands can be removed and further cleaned.
Such chemical synthesis works great for making really short pieces of DNA (like primers for PCR) but since more and more “drop out” each round because you have to terminate them because they failed to couple the yield (how much product you’ll get) drops off the longer you go (and there’s more chance of errors). So, the upper limit’s about 200 nucleotides. But more commonly you’re ordering things that are only 20-ish nucleotides long, frequently PCR primers. bit.ly/pcrprimer
Another way to get custom RNAs made is with in-vitro transcription, which uses a phage (bacteria-infecting virus) RNA polymerase to make template-based copies similarly to how it happens in our cells, but in this case you’re doing it in a test tube. It can be used for making longer RNAs, but your transcript has to start with letter(s) the polymerase likes (so, G if you’re using T7 RNAP). bit.ly/t7rnap
LOTS more on DNA & RNA bit.ly/nucleicacidstructure
more on oligonucleotides: blog: bit.ly/oligomeaning ; TH-cam: th-cam.com/video/W0WbdR1aj20/w-d-xo.html
more on ASOs: more on antisense oligonucleotides (ASOs): bit.ly/ASO_biochemistry & th-cam.com/video/KJGsTNmU_2M/w-d-xo.html
resources:
atdbio has a really nice guide: atdbio.com/nucleic-acids-book/Solid-phase-oligonucleotide-synthesis
and here’s one from Sigma on DNA Oligonucleotide Synthesis: www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/pcr/dna-oligonucleotide-synthesis
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com
Thank you for everything ❤️
you are GOAT never stop teaching
Thanks so much!
So how long does it take to make one of these synthetic DNA’s? 20 minutes?
No clue, sorry!
Thank you so much! *take my money* I'll join a Patreon if you have one.
That's very kind of you thank you! But I don't monetize anything.