In recent years, the extraordinary abilities of mRNA to induce potent immune responses against pathogens are publicly acknowledged. However, mRNA displays some other shortages including instability and limited translational capacity, which to some extent affect the effectiveness of mRNA vaccines.

Academia and some pharmaceutical corporations have tried to address this issue by incorporating modified nucleosides in mRNA, which offers advantages for the generation of potent and long-lived antibody responses. The so-called nucleoside modification is proved an effective method to reinforce mRNA stability, improve mRNA translation efficiency, and reduce activation of innate immunity.

PseudoUridine, also called the "fifth nucleotide" of RNA, is the most abundant modification in total cellular RNA. It's so prevalent that you can find it in rRNAs, tRNAs, and snRNAs. PseudoUridine is formed via isomerization of uridine, by binding the ribose C1 to uracil C5 instead of N1. Moreover, there are a great number of derivatives of pseudoUridine which can be acquired by N1 and/or N3 functionalization on uracil, or by deoxidation of ribose, or protection of hydroxyl groups on ribose.

Below this article, we will talk about several vital pseudoUridine derivatives that are of great help to researchers who are proceeding with mRNA vaccine research.

Phosphoramidite PseudoUridine

Phosphoramidites usually work as building blocks in the chemical synthesis of DNA/RNA oligonucleotides through 3'→5' direction. It can facilitate the introduction of pseudoUridine into RNA oligonucleotide and allow RNAs with single or multiple pseudoUridine modifications to be synthesized efficiently.

Currently, pseudoUridine-3'-phosphoramidite is in huge demand to produce stable RNA or DNA oligo. However, careful attention should be paid when applying them. In general, the effectiveness that pseudoUridine modifications exert on RNA stability and structure largely depends on their locations. Furthermore, when dealing with RNA phosphoramidites, it is indispensable to avoid degradation by exposure to RNase enzymes.

Phosphate PseudoUridine

Phosphate pseudoUridine can be divided into three categories: pseudouridine 5ʹ-monophosphate (ψMP), pseudouridine 5ʹ-diphosphate (ψDP), and pseudouridine 5ʹ-triphosphate (ψTP).

Over the years, researchers have been facing the plight that mRNA therapy development and application are always restricted by the immune toxicity in the host. Thanks to the mRNA modification utilizing naturally occurring modified nucleotides like pseudouridine-5'-triphosphate, the immune toxicity problem is dramatically alleviated. The biological applications of other phosphate pseudoUridines are still under investigation. Hopefully, more useful tools for gene replacement and vaccination will be explored in the near future.

Modified PseudoUridine

Some modifications exist in RNAs naturally, which are also called derivatives of pseudoUridine. Scientists have found 4 natural RNAs modification, namely 1-methylpseudouridine (m1ψ), 3-methylpseudouridine (m3ψ), 2'-O-methylpseudouridine (ψm), and a hypermodified residue 1-methyl-3-(3-amino-3-carboxypropyl) pseudouridine (m1acp3ψ). 

As for the function of these modified pseudoUridines, m1ψis an outstanding representative with a brilliant performance to boost protein expression but without cytotoxicity at different concentrations. The application of m3ψ will also benefit the thermodynamic stability of the RNA, which even outperforms pseudoUridine.

Most of the modified pseudoUridines derive from the hydroxy group on ribose or amino in the uracil base of pseudoUridine. Additionally, chemical synthesis provides advancements in their function. As revealed in some authoritative journals, the chemically synthesized 4-thiopseudouridine with thiocarbonyl groups will stabilize the triplex formation of triplex-forming oligodeoxynucleotides (TFOs).

Conclusion

At the moment when the world is in urgent need of the prevention and treatment of COVID-19, the whole pharma industry is striving for valid solutions to improve stability and efficacy of mRNA vaccines. It turns out these efforts are not in vain and at least an increasing number of pseudoUridine and its derivatives are already employed to optimize mRNA property and structure.