Pfizer developed a technique for determining the primary structure of their mRNA vaccine including detecting base modifications, the 5'-cap, and the poly-A tail

Two of the most widely used vaccines during the pandemic were based on mRNA technology.

Messenger ribonucleic acid (mRNA) is the template that's used by our cellular machinery to create proteins.

Traditionally, vaccines are made using killed viruses, weakened viruses, or viral proteins that have been produced synthetically.

These all make great vaccines, but creating vaccines this way is time consuming, and that's something that's in short supply during a pandemic!

This is where mRNA comes in and this technology leverages decades of research on how best to express proteins directly in cells, and in the case of a vaccine, present those proteins for recognition by the immune system.

Getting mRNA into cells is relatively easy.

But that's when the problems start because our cells are very sensitive to RNA and they chop up anything they think is foreign as a defense mechanism.

Thankfully, it was discovered that if you replace uracil in the mRNA with a modified base, N1-methyl-pseudouridine, cells didn't sound the invasion alarm.

But that's not the whole story because for an mRNA to be stable and translated into protein, it must be 5'-methylated or capped, and also have a Poly Adenosine (Poly-A) tail.

So, for an mRNA vaccine to work, it needs to be capped, have a poly-A tail and be full of modified uracils.

How are we sure that the mRNA in the vaccine has all of these things?

I imagine that in the fall of 2020 a series of emails were exchanged with the FDA.

FDA: What quality control have you done on the primary structure of the RNA to be sure it's translation competent?

Pfizer: We sequenced it, it's fine.

FDA: Thank you for your reply. Is the cap intact? What about the poly-A tail? And you used N1-methyl-pseudouridine instead of uridine (uracil), how do you know it's N1-methyl-pseudouridine in there?

Pfizer: ...

At least this is how I'm guessing it all went down.

So Pfizer had to figure out a way to show that everything about the primary structure was correct. Not just the sequence but that the sequence also contained all of the appropriate modifications.

They decided to do this with mass spectrometry because it can be used to determine the chemical makeup of molecules in a solution.

It does this by ionizing the molecules and then smashing them into a detector to determine their mass to charge ratio (pretty cool!)

And so that brings us to the figure below.

Here Pfizer chopped up their vaccine mRNA, threw it on a mass spec, and looked at the masses of everything that came out making sure they detected all of the sequence features including the methylated cap and the poly-A tail!

This is an ingenious way to determine the complete primary structure of a complex nucleic acid beyond just sequencing its bases!