Bert Hubert recently published Reverse Engineering the source code of the BioNTech/Pfizer SARS-CoV-2 Vaccine and I (along with the rest of the internet) loved it! It provided a clear review of Pfizer’s mRNA vaccine written for a software engineering audience. Given my previous synthetic biology experience, I was excited to read about how their vaccine actually worked.

Bert linked to a document by the WHO containing the genetic sequence of the mRNA. In that sequence, the symbol “Ψ” represents 1-methyl-3′-pseudouridylyl. Bert explained that this was a clever substitution to help the mRNA escape our immune system’s detection. He also linked to a tweet about Karikó and Weissman which implied that they had discovered 1-methyl-3′-pseudouridylyl.

I was fascinated! I’d never heard of Ψ before. Digging in a little deeper I realized that the symbolic representation had confused me, and maybe other folks were confused too.

Karikó and Weissman published a lot of papers about pseudouridine, which they denoted with “Ψ.” Replacing uracil for pseudouridine in mRNA does indeed help the mRNA escape our immune system’s detection [1]. However, pseudouridine is not pseudouridylyl.

Looking at the WHO document that Bert linked, the two molecules are structurally similar but definitely not the same. I’m not any good at organic chemistry, but my guess is the extra phosphorus and oxygens are from the sugar phosphate backbone. But what’s with the extra methyl group (carbons and hydrogens) on the nitrogen? My intuition says that’s important.

pseudouridine 1-methyl-3′-pseudouridylyl
Wikipedia WHO 11889

So what’s pseudouridylyl?

Why did Pfizer choose to use pseudouridylyl instead of pseudouridine? I had guessed that it must offer either a biological benefit (increasing effectiveness) or a manufacturing benefit (reducing cost). “Pseudouridylyl” (or even “uridylyl”) is not a common word either on the internet or in the literature. I found lots of references to similar chemicals, but couldn’t find literature linking any of them to immune system responses or mRNA synthesis.

And then came the a-ha moment!

In the WHO document, the author represented 1-methyl-3′-pseudouridylyl as “m1Ψ” (with “m1” indicating a modification). However, in the actual genetic sequence, the author chose to abbreviate “m1Ψ” to simply “Ψ.”

Now that I knew what I was looking for, I found an abundance of papers referencing m1Ψ. The canonical name seems to be “N(1)-methylpseudouridine.”

N(1)-methylpseudouridine looks to be an even better substitution than pseudouridine [2]. It also shares the convenient property of escaping immune system detection [3]. The N(1)-methylpseudouridine papers definitely build upon the work of Karikó and Weissman.

The diagrams show that they’re pretty close! See the methyl group?

N1-Methylpseudouridine-UTP 1-methylpseudouridine 1-methyl-3′-pseudouridylyl
Jena Bioscience Modomics WHO 11889

I’m now fairly confident that Ψ in the Pfizer sequence is m1Ψ. They’re calling it “1-methyl-3′-pseudouridylyl,” but it’s also known as “N(1)-methylpseudouridine” or “1-methylpseudouridine.”

References

[1] Karikó K, Muramatsu H, Welsh FA, Ludwig J, Kato H, Akira S, Weissman D. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther. 2008 Nov;16(11):1833-40. doi: 10.1038/mt.2008.200. Epub 2008 Sep 16. PMID: 18797453; PMCID: PMC2775451.

[2] Callum J C Parr, Shunsuke Wada, Kenjiro Kotake, Shigetoshi Kameda, Satoshi Matsuura, Souhei Sakashita, Soyoung Park, Hiroshi Sugiyama, Yi Kuang, Hirohide Saito, N1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells, Nucleic Acids Research, Volume 48, Issue 6, 06 April 2020, Page e35, https://doi.org/10.1093/nar/gkaa070

[3] Andries O, Mc Cafferty S, De Smedt SC, Weiss R, Sanders NN, Kitada T. N(1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. J Control Release. 2015 Nov 10;217:337-44. doi: 10.1016/j.jconrel.2015.08.051. Epub 2015 Sep 3. PMID: 26342664.

Suppose you had sales table with timestamp and customer_id as the columns.

id timestamp customer_id
1 2011-03-17 10:31:01 7
2 2011-03-17 10:45:05 8
3 2011-03-17 10:47:05 7
4 2011-03-17 10:50:10 8

You want to query for what’s the average time between purchases for each customer.

You can use:

SELECT t.customer_id,
       AVG(t.difference) as interval
FROM ( 
  SELECT customer_id,
         timestamp - lag(timestamp) OVER (PARTITION BY customer_id ORDER BY timestamp) AS difference
  FROM sales
) t
GROUP BY t.customer_id

You’ll get:

customer_id interval
7 00:16:04
8 00:05:05

Sources:

Here are the steps I use to cast a fish keycap. If you’re completely new, see the Z-Butt wiki and my older blog post.

petri fish at an angle

First I cast a bottom piece. Thanks to the Z-Butt community, I learned to get the petri effect, you need to wait for about 1-2 hours before adding in alcohol ink to the resin. Here I waited for 1.5 hours.

alcohol ink

After the bottom piece is cured, I add a polymer clay fish. I use acrylic paint to add details and dab a dollop of resin to secure the fish to the base. When everything looks dry, I invert it to cast the top piece.

WIP keycap

That’s it!

After casting keycaps over a couple of months, I’ve finally finished my first bottle of resin. Now I’m trying out a different brand and realize not all epoxy resins have the same properties. I started with Unicone Art a couple of months ago and now am using Dr Crafty.

Here’s a comparison of the resin brands Unicone Art (left) and Dr Crafty (right):

resin compare

Epoxy Resin Brand Unicone Art Dr Crafty
Cure Time 18 hours – hard enough to work with, 24+ hours – full hardness 24 hours – still very pliable, 30 hours – hard enough to work with
Casting Great for casting More viscous and will not conform to certain complex molds
Clarity Clear but small trapped air bubbles will make the keycaps appear cloudy Crystal clear and fewer air bubbles
Odor I couldn’t detect anything Strong chemical smell from the hardener

If you look closely at the photo above (and ignore the different dye colors), there are a lot more tiny air bubbles in the left keycap than the right. I let all resin degas before casting, but it seems like for Unicone Art, the air bubbles have a harder time making their way out.

I prefer strongly Unicone Art for its casting property and turnaround time. However, Dr Crafty is visibly clearer.

Maybe I’ll try to cast the bottom pieces with Unicone Art and the clear top with Dr Crafty. Will report back.