Complexities Surrounding N-1-Methylpseudouridine (m1Ψ) in

COVID-19 Synthetic Gene Therapies

Andrew Zywiec1, Abdul Mannan Baig2

N-1-methylpseudouridine (m1Ψ) was introduced the misreading of near-cognate stop codons or in-
into modified-mRNA vaccines to reduce immuno- crease the chance of the ribosome dissociating from
genicity and increase mRNA stability, but its pres- the mRNA before reaching the natural stop codon.
ence can indeed affect translation in several ways. This dynamic potentially leads to the issue of ribo-
(1) Uridine in mRNA pairs with adenosine in tRNA. somal slippage, stalling, or both. (4) The ribosome
However, m1Ψ, although structurally similar to ur- might pause at sites containing m1Ψ due to altered
idine, can alter base-pairing dynamics. The dynam- codon-anticodon interactions or because m1Ψ can
ics of mRNA and tRNA interaction are affected by change the mRNA's secondary structure, affecting
anything that alters the temporal, structural, or bio- how the ribosome moves along the mRNA. Riboso-
chemical nature of the assembly and its apparatus. mal pausing or altered codon-anticodon interactions
(2) This may lead to reduced efficiency of codon can increase the chance of +1 or -1 frameshifts due
recognition, as the tRNA's anticodon may not pair to ribosomal slippage, especially at slippery se-
as efficiently with m1Ψ as it would with uridine. quences or if the mRNA structure is altered by
This potentially leads to misreading. If the modifi- m1Ψ, affecting the reading frame for all subsequent
cation affects base-pairing enough, it might increase codons until a stop codon is encountered or the
the chance of a tRNA with a noncognate anticodon mRNA ends. (5)
binding, leading to amino acid misincorporation. (3)
Example:
This concern is significant. The alteration of one
amino acid can have significant consequences, as is Original mRNA sequence: AUG GCU ΨGG UAA
the case in sickle cell anemia. At the 6 position, a GCA CCC UGU UAG
glutamic acid (hydrophilic) is traded for a valine Slippage at Ψ sequence: AUG GCU GGU AAG
(hydrophobic), which alters the tertiary structure of
CAC CCU GUU AG . . .
the protein such that it causes profound disease. If
ribosomal movement is disrupted, it might allow for The Pfizer-BioNTech (BNT162b2) spike protein
coding sequence in this vaccine has 728 uridines re-
Keywords: Pseudouridine, mRNA, translation placed by m1Ψ. (6) Similarly, the Moderna vac-
cine's spike protein mRNA also has its uridines re-
placed with m1Ψ. (7) Although exact counts might
slightly differ based on specific codon optimization
or slight sequence variations, the number is in the
same range. Ignoring all other possible concerns
above (misincorporation, dissociation, etc.), let us
1.
focus solely on the possible aberrant proteins cre-
Ekklesia Research Group
2. ated by this positive or negative frameshift:
St. Georg Hospital, Bad Abiling, Germany
Each m1Ψ can cause a frameshift: We'll consider
that any one of the 728 m1Ψ sites could inde-
pendently lead to either a +1 or -1 frameshift. There-
Corresponding author: fore, each m1Ψ site can result in three outcomes: no
Andrew Zywiec, MD, CTP change, +1 frameshift, or -1 frameshift. This means
Ekklesia Research Group the potential number of distinct proteins possibly
1030 Shoreview Circle #206, Casselberry, FL 32707, USA created is:
Tel: 440 319 7422
Email: [email protected] 3728 = 2.67 × 10346

J Indep Med 2025 Vol. 1 No. 2 https://doi.org/10.71189/JIM/2025/V01N02A11 171

For reference, there are ~1082 atoms in the known folded proteins, which can aggregate, leading to cel-
universe. (8) lular stress, toxicity, or contributing to neurodegen-
erative disorders like prion diseases. (11) Also, an
This represents the theoretical maximum of unique
influx of misfolded or aberrant proteins might over-
protein sequences from frameshift mutations at
whelm cellular degradation pathways like the ubiq-
every m1Ψ site. The actual diversity of proteins pro-
uitin-proteasome system or autophagy, leading to
duced would be constrained by cellular mechanisms
cellular dysfunction. Likewise, this could over-
like nonsense-mediated decay, no-go decay, and ri-
whelm cellular energetics and cause mitochondrial
bosome-associated quality control. Further, it is sta-
dysfunction. (12) While unlikely, functional pro-
tistically improbable for the majority of these sce-
teins could be resultant of this process, with un-
narios. However, even with these constraints, the
known/unintended consequences. For example, if
potential for variation is significant, illustrating the
these proteins could interact with chromatin or af-
complexity of mRNA translation when considering
fect gene expression, they could lead to epigenetic
modifications like m1Ψ.
modifications with long-term implications for cell
There are a host of potential concerns surrounding function or lineage, or if they can interact with the
this issue of aberrant protein production. First, an cell cycle, they could promote tumorigenesis. (13)
unintended protein sequence could be recognized The long-term presence of these proteins could have
by the immune system as foreign, potentially lead- drastic implications, especially if they interfere with
ing to an inflammatory response. (9) If these pro- normal cellular processes. This is a very limited dis-
teins share homologous sequences with human pro- cussion of the topic, and is by no means exhaustive
teins, there's a risk of triggering autoimmunity or comprehensive, and I hope it serves to invite fur-
where the immune system attacks the body's own ther and very necessary discussion.
tissues. (10) Additionally, this could lead to mis-
.

172 https://doi.org/10.71189/JIM/2025/V01N02A11 J Indep Med 2025 Vol. 1 No. 2

References
1. Karikó K, Muramatsu H., Welsh FA, et al. In- Covid-19 vaccine. N Engl J Med. 2020;383(27):
corporation of pseudouridine into mRNA yields 2603–2615. doi:10.1056/NEJMoa2034577
superior nonimmunogenic vector with in- 7. Anderson EJ, Rouphael NG, Widge AT, et al.
creased translational capacity and biological Safety and immunogenicity of SARS-CoV-2
stability. Mol Ther. 2008;16(11):1833–1840. mRNA-1273 vaccine in older adults. N Engl J
doi:10.1038/mt.2008.200 Med. 2020;383(25):2427–2438. doi:10.1056/
2. Rodnina, MV, & Wintermeyer W. Fidelity of NEJMoa2028436
aminoacyl-tRNA selection on the ribosome: ki- 8. Tegmark M. Precision cosmology. Phys Today.
netic and structural mechanisms. Annu Rev Bi- 2006;59(4):36–41. doi:10.1063/1.2207037
ochem. 2001;70:415–435. doi:10.1146/an- 9. Gallucci S, Matzinger P. Danger signals: SOS
nurev.biochem.70.1.415 to the immune system. Curr Opin Immunol
3. Grosjean H, de Crécy-Lagard V, Marck C. 2001;13(1):114–119. doi:10.1016/S0952-7915
(2010). Deciphering the complex world of (00)00191-6
tRNA modifications: the role of modifications 10. Oldstone MBA. Molecular mimicry and
in decoding and translational fidelity. FEBS immune-mediated diseases. FASEB J. 1998;
Lett. 2010;584(2):252–264. doi:10.1016/j. 12(13):1255–1265. doi:10.1096/fasebj.12.13.
febslet.2009.11.062 1255
4. Drummond DA, Wilke CO. The evolutionary 11. Soto C, Pritzkow S. Protein misfolding, aggre-
consequences of erroneous protein synthesis. gation, and conformational strains in neuro-
Nat Rev Genet. 2009;10(10):715–724. doi:10. degenerative diseases. Nat Rev Neurosci.
1038/nrg2662 2018;19(10):652–665. doi:10.1038/s41583-
5. Atkins JF, Björk GR. A gripping tale of 018-0059-3
ribosomal frameshifting: extragenic suppress- 12. Hipp MS, Park SH, Hartl FU. Proteostasis
ors of frameshift mutations spotlight P-site impairment in protein-misfolding and aggre-
wobble. Cold Spring Harb Perspect Biol. gation diseases. Trends Cell Biol. 2014;24(9):
2009;1(4):a001859. 506–514. doi:10.1016/j.tcb.2014.05.003
doi:10.1101/cshperspect.a001859 13. Hanahan D, Weinberg RA. Hallmarks of
6. Polack FP, Thomas SJ, Kitchin N, et al. (2020). cancer: the next generation. Cell. 2011;144(5):
"Safety and efficacy of the BNT162b2 mRNA 646–674. doi:10.1016/j.cell.2011.02.013
.

J Indep Med 2025 Vol. 1 No. 2 https://doi.org/10.71189/JIM/2025/V01N02A11 173

 This page is intentionally left blank

174 https://doi.org/10.71189/JIM/2025/V01N02A11 J Indep Med 2025 Vol. 1 No. 2