Brief history of pseudouridine and mRNA vaccines

After reading Bert Hubert’s Reverse Engineering the source code of the BioNTech/Pfizer SARS-CoV-2 Vaccine and figuring out what 1-methyl-3′-pseudouridylyl was, I ended up reading a lot of papers.

Since I was a very junior biologist and am not familiar with this area of biology, I didn’t know really know where to start. I mostly followed headlines and citations. Bert provided a great jumping off point by referencing Karikó and Weissman. Their story is fascinating.

In 1998, Karikó wrote a paper on a method to more efficiently deliver DNA or mRNA to cells (Phosphate-enhanced transfection of cationic lipid-complexed mRNA and plasmid DNA). This method doesn’t involve any uracil substitutions and instead modifies the chemical mixture containing the DNA and mRNA before the delivery process.This doesn’t do much in itself, but they used these protocols to speed up their later research.

In 2005, Karikó and Weissman published a paper on substituting uracil for pseudouridine in mRNA. The modified mRNA doesn’t trigger Toll-like receptors (Suppression of RNA Recognition by Toll-like Receptors: The Impact of Nucleoside Modification and the Evolutionary Origin of RNA). Toll-like receptors are proteins primarily expressed by cells that are a part of the immune system, so the immune system doesn’t detect the modified mRNA and therefore doesn’t destroy it.

To put this in software engineering terms, imagine a firewall (the immune system) with a rule (the Toll-like receptor) examining incoming requests (the mRNA) and blocking any with a certain parameter (mRNA containing uracil). Modifying the mRNA by replacing the uracil with pseudouridine is the equivalent of dropping that parameter so that the request passes that rule. The firewall keeps working, and other rules may still block the request, but at least it’s passed that one.

In 2008, Karikó and Weissman discovered that by substituting pseudouridine in mRNA they could reduce the immune system response and simultaneously make the mRNA more effective at expressing the protein it encodes (Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability).

In our software metaphor, that’s analogous to passing all of the firewall rules!*

This is a really exciting discovery for mRNA vaccines!

I was surprised to learn that pseudouridine sometimes replaces uracil in “natural” mRNA. I’d thought of RNA as being composed of adenine, cytosine, guanine, and uracil, but researchers have known since 1951 that pseudouridine is also a nucleobase (the actual paper mentioning this is Nucleoside-5′-Phosphates from Ribonucleic Acid but I couldn’t find a copy so I’m relying on a citation from Pseudouridine Formation, the Most Common Transglycosylation in RNA).

The early discovery of pseudouridine and later discovery of its usefulness in mRNA vaccines reminds me a lot of the CRISPR discoveries. CRISPR was discovered as a bacterial immune system feature in 1987, but Doudna and Charpentier leveraged it as a powerful DNA editing tool in 2011 and won the Nobel for it this year. Researchers stand on the shoulders of giants!

If you’re interested in a more technical history of pseudouridine, Pseudouridine in RNA: What, Where, How, and Why is a good summary from 2008.

* In biology, very few things happen 100% of the time. Since biological systems are often stochastic, reactions are measured by percent effectiveness compared to something else. I’m mentioning this here so you don’t worry that we have no immune defenses against mRNA bioweapons and the end is nigh.

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