Phosphoramidite is pretty cool – am I right or am I right? It helps us DNA or RNA write! Solid-state oligonucleotide synthesis, including the phosphoramidite method, can be used by companies to make large amounts of short DNA or RNA sequences (oligonucleotides or “oligos”). So I thought I’d do you a solid and tell you about what might happen when you order your PCR primers!

It’s mother/daughter bond-ing time, so yesterday I told you about how my mom and I made peptide bond models. Peptide bonds are the kind of bond that connect protein letters (amino acids) – and we made our models out of clay and wire – so not very functional. But scientists can make fully functional DNA & RNA chains, complete with their usual phosphodiester bonds between nucleotides (DNA or RNA letters), but they do it kinda “backwards.” But I got ahead of myself, let me step back and start from the beginning… 

“Nucleic acids” include DNA & RNA and they’re made up of nucleotide letters.Nucleotides are made up of of a sugar (ribose in RNA & deoxyribose in DNA) connected to a unique nitrogenous base (A, C, G, or U (or T in DNA)) and phosphate(s). If you don’t have the phosphate(s), you call it a nucleoSide. There’s a big difference between how nucleotides are added in cells and how they’re added through chemical synthesis…

Your cells can only copy existing DNA (into DNA or RNA) because they rely on an existing template. When using solid-state synthesis methods to custom-make short pieces of DNA or RNA (oligonucleotides or “oligos”), there is no template, you just introduce letters one at a time, so you can write “anything.” But you have to tightly control it because, since there’s no template there’s no way to “proofread” – so you have to make sure excess letters don’t linger – and you have to make sure that the letters get linked correctly – in your cells, proteins ensure this specificity because when they hold the letters in addition position only the right way is available. 

It involves multiple steps and some longish chemical names but just like most chemical synthesis reactions, at its heart, the logic’s “simple” -> hide what you don’t want changed (PROTECT) & make what you do want changed more easily changeable (SELECTIVELY DEPROTECT) -> add something to change it (COUPLE) -> remove unreacted things (CAP) -> repeat cycle until you’re satisfied -> remove any “hiders” you don’t want in your final product (FULLY DEPROTECT) -> clean it up (DESALT, HPLC/PAGE purify, etc.)

Similar methods can be used for DNA & RNA, but if you want RNA, it’s a lot more $ you’ll have to pay! DNA & RNA differ by a single oxygen (that D’s for “deoxy”), yet custom DNA’s cheap, but RNA’s expensive! It’s not just because RNA’s cooler (though it is) or because oxygen’s super pricey, it’s what that oxygen can do… it likes to kick off its neighbors, breaking the chain of letters being written or stealing it for itself… So when companies synthesize RNA using SOLID-STATE OLIGONUCLEOTIDE SYNTHESIS, they have to protect the RNA from itself – and protect their profit margins…

We use numbers with a “prime” sign (‘) to indicate where things stick off of the sugar – I like to imagine nucleosides as little people with the left arm being the 5’, right arm 1’, right leg 2’ and left leg 3’. The difference between RNA & DNA (other than that U vs T thing) is that RNA has another OH as its “right leg” whereas DNA just has a hydrogen (H) which often isn’t even drawn in, just implied.

What difference could an oxygen make? A LOT! In solid-state oligo synthesis, you chemically hide (block/protect) or “unhide” (deprotect) these arms & legs to direct how they link up. But in RNA you have to “boot” 2 legs, not just one, and you have to keep one of those boots on while you remove the other.  Because that “right leg” can kick off the growing chain or steal it from the left!

In the form of nucleosides your cells use, a phosphate (PO4-) “hand” is attached at the 5’ arm & there’s a hydroxyl (-OH) foot at the 3’ leg. In cells, like the bacteria we use to amplify plasmids, or in PCR, a POLYMERASE protein helps link nucleotides together by forming phosphodiester bonds between the phosphate of 1 nucleotide (attached at the 5’ location on the sugar) & the 3’ -OH of the next.

Chemical synthesis instead adds them as modified “nucleoside phosphoramidites” – instead of having the phosphate group come from the “left arm” (5’) it comes from the “left leg” (3’). And they’re added “right to left” 3’->5’ but the end products are the same. 

note: an important consequence of this is that the beginning of your DNA or RNA oligo will have a 5’OH instead of 5’ phosphate(s) unless you pay them more to add it on

That’s not the only difference – since you don’t have a polymerase relying on a template, you have to direct the show & make sure things go where they’re supposed to.

For both DNA & RNA you need “gloves”  – a DMT (4,4’-dimethoxytrityl) group hiding the “left arm” and things like benzoyl or isobutyryl groups to protect the “right arm” (bases) because the same atomic arrangements that allow bases to bind to one another also allow them to bind to things you don’t want. And both DNA & RNA need a “sandal” on their left leg – a 2-cyanoethyl-diisopropylamino group partially protecting the phosphate group of the 3’ leg so it doesn’t “overreact” but leaves it available for the oxygen from the next letter to attack it

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. http://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). http://bit.ly/t7rnap 

LOTS more on DNA & RNA http://bit.ly/nucleicacidstructure 

_more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0

 

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