Why are vaccine mRNAs chemically altered?

One of the confusing aspects of the mRNA vaccines, and therefore somewhat mysterious, is that the genetic material used to construct the mRNA uses chemical nucleotide bases that are not typical constituents of natural mRNA genetic material (we will get into the background of all this right away). These are pseudouridines, or more correctly, N1-methyl-pseudouridine. A somewhat cryptic-looking symbol Ψ also denotes this chemically modified nucleotide base for pseudouridine and m1Ψ for N1-methyl-pseudouridine.

The reasons given why the vaccinal mRNA is chemically altered as such are at least twofold, and currently denote the pinnacle of biomolecular technological achievements to finally allow the delivery of mRNA into humans for genetic manipulation. These are:

• To dramatically reduce innate immune system’s activation against foreign mRNA that is being injected into the body;
• To increase the use of mRNA as a template for the spike protein production inside the cells that take up the foreign genetic material by being more resistant to degradation;
• To possibly increase the efficiency of the use of mRNA during protein production by the cellular machinery that uses mRNA templates to produce proteins.

With these new mRNA vaccines, for the first time ever, we have been able to conduct an experiment of genetic manipulation (on a yet unknown scale) by being able to manipulate how the body’s cells are forced to use mRNA templates. The potential of this technology for the future is enormous in being able to deal with different human diseases (or infection threats as in the case of COVID-19 vaccines) by simply modulating how proteins are made in the body. But we have to learn lots of tiny details along the way for us to understand how such technology can be fine tuned for minimal hazards and the greatest desired outcomes.

But first, as promised…

Quick genetics background

Genetic material is made up of four chemical bases, and there are two types of genetic material: DNA and RNA. DNA is primarily a repository of information. RNA are tiny copies of DNA that are used to regulate the function of the cell by manipulating how proteins are made and used. Proteins, on the other hand, are tiny miniature molecular robots (and to some degree RNA molecules can also be tiny functional robots) that perform all the tasks in the cell that provide the final illusion that this collection of chemicals is actually alive (and how it springs into life).

DNA and RNA are almost identical with a small chemical difference between the DNA and RNA. In DNA, the four chemical bases used in its production are adenine, cytosine, guanine, and thymine, which is where we get the famous abbreviations A, C, G, and T that make up the DNA code. It is the arrangements of these four chemicals that make the code. These chemicals are attached to sugar molecules (called ribose – not like the sugar you eat though) and it is these sugar molecules linked together that make up the backbone of the DNA with the A, C, G or T chemical portions sticking out to interact with one another (where A can bond with T and G can pair with C through electrostatic interactions – for sake of simplicity, think of it like a magnetic attraction towards each other). This is how you can have two strands of DNA form a complementary double-stranded helix with two identical DNA strands facing and bound to each other in reverse order, and also how DNA can be used to produce more copies of itself.

The sugar backbone to which the nucleotide bases are attached is what is different between the DNA and RNA, with DNA having one hydroxyl group (oxygen atom bound to a hydrogen atom) removed in comparison to the sugar in RNA backbone and hence you get the DNA name: deoxy-(indicating the removed hydroxyl group)-ribo-(ribose is the sugar used as a backbone)-nucleic-acid-(indicates that numerous nucleotides are linked together). RNA is ribo-nucleic-acid.

Besides the sugar backbone, there is another major difference between DNA and RNA. DNA uses thymine, whereas RNA uses a very closely related chemical called uracil (or uridine once coupled to the sugar backbone). Just like thymine, uridine of RNA can still chemically interact with adenine.

We now finally get to pseudouridine, which is like uridine that is further chemically modified. The difference is subtle, as only the position of two atoms is reversed. Otherwise, they look the same. That small switch gives pseudouridine unique properties. It also exposes pseudouridine to further chemical modification (attachment of a few more atoms to the existing chemical arrangement of atoms in space), to produce N1-methyl-pseudoruridine that is used the in the COVID-19 mRNA vaccines. In this case, a carbon atom bound to few hydrogen atoms (referred to as methyl) is attached in a specific spot of pseudouridine. This last modification makes the N1-methyl-pseudouridine resemble the thymine that is used in DNA. In the current COVID-19 mRNA vaccines from both Pfizer-BioNTech and Moderna, every uridine in the mRNA coding for the spike protein was substituted with N1-methyl-pseudouridine.

All these modifications do not modify the ability to chemically interact with the adenine nucleotide, thus uridine and pseudouridine or N1-methyl-pseudouridine all can interact with adenine, thus in theory their use is undistinguishable in terms of chemical interaction with genetic code nucleotides.

One of the most common misconceptions associated with the use of pseudouridines is the assumption that they are not natural, and therefore the genetic material used in vaccines is synthetically made with components not found in nature. That is not correct, pseudouridines apparently can encompass ∼0.2–0.6% of the cell mRNAs sequences. Even N1-methyl-pseudouridine is used in the modification of RNAs other than mRNAs while whether or not it is also a natural modification of mRNAs seems to be yet determined.

What happens in the wild?

However, the overall biological significance of mRNA pseudouridination itself definitely remains unclear. This is such newfound knowledge that we simply have to face the ugly gaps at this moment. This means, there is lots yet to be discovered and understood about pseudouridine and N1-methyl-pseudouridine. To give you a sense of how new this information is, the first existence of pseudouridine being confirmed as part of mRNA produced by human cells was only in 2015. Thus, our awareness of this was born very, very recently.

The same study that identified the first mRNA content amount, also showed that the use of pseudouridine in mRNAs can be a highly dynamic process, in response to different environmental conditions. The authors even comment that it is tempting to imagine such rapid use of pseudouridines in response to environmental changes actually taking place in nature, including the existence and activation of molecular machinery that can change the levels of pseudouridine in mRNAs.

Why might these modifications be happening in nature?

One emerging theory is to possibly increase the diversity of produced proteins in response to environmental stress.

The most immediate impact of any RNA modification is on the modified RNA molecule itself. You can almost think of it where any chemical modification of RNA (or DNA for that matter) affects the lifespan of that molecule in one way or another. There will be some biological consequences, likely starting with the altered lifespan of the modified RNA itself. But chemical modification can also influence many different steps involved in the RNA’s molecular use.

What are the consequences of introducing a high quantity of chemically modified nucleotide bases into human circulation? Can it have any specific impact on how the spike protein is made using such modified mRNA as a production template? Can any of these modified nucleotides be reused by the body?

It is at least possible that the product of modified mRNA could be different than anticipated. One recent preprint (see the previous post for preprint definition) suggested that the use of either pseudouridines or their methylated counterpart, 1-methylpseudouridines, can influence how likely it is that incorrect amino acid building blocks could be used in the production of the protein. Could this happen during the production of spike proteins from mRNA vaccine templates?

Accordingly, modified nucleotides in the vaccine mRNA could possibly have the ability to introduce incorrect amino acids in the spike protein. It appears this can occur during the use of the modified mRNA as a template to produce proteins. Proteins are built with amino acids, and special RNA molecules that are carriers of amino acids (these are called tRNAs) are selected based on the genetic code complementarity we talked about above. Hence the mRNA code is used as a template to select specific tRNAs to determine which amino acids are going to be linked together during the production of a protein. The genetic code on the mRNA is referred to as a codon, and the matching genetic sequence of tRNA is called an anti-codon. This process ends up building an entire protein, which is made of many different amino acids linked together in one long chain. Along the protein production process, this long chain of amino acids adopts a complex three-dimensional shape. Often, the final protein product doing the cellular work is an assembly of multiple proteins, sometimes the same proteins, sometimes many different types of proteins. The spike protein itself is composed of three identical protein units that come together to provide the final shape.

Modified nucleotides, depending on the surrounding genetic code sequence, can allow interaction with incorrect tRNAs which will bring incorrect amino acids into the incorporation of the growing chain of amino acids that will make up the future final protein.

In other words, modified RNAs can lead to mutations in the expected protein product.

Interestingly, the authors of that preprint, comment that this actually could be an advantage for vaccines. Introducing the mutations in some of the protein spike proteins would result in a broader range of very similar but slightly different spike proteins which could result in a greater diversity of antibodies produced against slightly different spike proteins. This could provide greater protection against the virus that is constantly mutating. This is similar to the expected increased efficacy of multivalent mRNA vaccines in comparison to the original variant mRNA vaccines. However, the authors do warn that the modified sequence composition “may in some cases prove to be critical to designing safe and effective mRNA therapeutics.”

This proposal that mRNA vaccines might have been helped in their efficacy by the modified nature of the genetic content is supported by a surprising finding of the efficacy of unmodified mRNA vaccine which was also tested. When the mRNA vaccine was developed with unmodified mRNA, but delivered with the same lipid nanoparticle that was used as a delivery method for the Pfizer-BioNTech mRNA vaccine, the unmodified mRNA vaccine efficacy against the symptomatic disease was only 48% in comparison to the high 90s for the modified mRNA vaccines from Pfizer-BioNTech and Moderna.

However, another study that specifically analyzed the production of spike protein from either modified or unmodified RNA template did not find the production of miscoded spike protein due to the use of modified mRNA. However, the authors noted that being able to determine this with high precision with the technology they used is challenging and low levels of miscoded proteins might simply have been missed due to the lack of detection sensitivity of technology. But it was clear to the authors that N1-methyl-pseudouridine behaved better in minimizing mutational outcomes than pseudouridine itself, which is some news of comfort.

The last point to address in the knowledge gap is to figure out how modified mRNA influences the overall genetic use of cellular mRNAs in the cell. Everything inside our cells works within a certain balance and biological processes can go astray when that balance is not maintained. This can also be said of how mRNAs are used in the cell. One of the ways how modified mRNAs achieve lower immune system recognition and escape degradation is by affecting the use of the specific mRNAs that code for different immune system proteins. But this still leaves us with the big question of how many other mRNAs are affected in their use by the cell?

To conclude, we are using a brand-new technology, that has never been used in humans before -– with its rapid output achieved through the incredible (but very recent) advances in how the mRNA genetic material can be delivered into a body and have enough time to produce the expected protein products before being destroyed. While we have crossed these amazing technological hurdles in terms of the manipulation of the mRNA as well as manipulation of the lipid vehicles used to deliver such mRNA (plot for a different post), we still have yet to learn much about how these constructs affect and influence our overall biology and what might be the possible consequences of such unknown biological influences.

Changing uridines for N1-methyl-pseudouridines is also not the only modification of mRNA coding for the spike protein either. Remember the concept of codons and how they can determine which tRNA can interact with mRNA during the production of the protein? There are different codons for the same amino acid carrying tRNAs, meaning there is a redundancy in the genetic code of mRNA in determining which amino acid is selected. Multiple different ways exist to select the same amino acid in the protein sequence. The COVID-19 mRNA vaccines have certain codons altered from what is naturally found in the SARS-CoV-2 virus code even though it leads to the insertion of same amino acid in the sequence. This is referred to as being codon-optimized and this is also a plot for another post. But as a consequence, the genetic sequence of COVID-19 mRNA vaccines is not exactly the same as that found in the virus, which means there could be unintended biological impacts although the assumption based on prior lab studies is that it should be a safe approach. We should celebrate the future potential of this powerful new technology but still take care to tread carefully with many deep analyses to ensure we gain the powerful insights necessary to safely control all the effects of this newfound tool of genetic manipulation.

This article has been produced by Merogenomics Inc. and edited by Jason Chouinard, B.Sc. Reproduction and reuse of any portion of this content requires Merogenomics Inc. permission and source acknowledgment. It is your responsibility to obtain additional permissions from the third party owners that might be cited by Merogenomics Inc. Merogenomics Inc. disclaims any responsibility for any use you make of content owned by third parties without their permission.

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