Detailed Dissection and Critical Evaluation of the Pfizer/BioNTech and Moderna mRNA Vaccines

In the paper

“First, while the -3R (site 4 in the first column of Table 5) has been demonstrated repeatedly to enhance translation initiation, the effect of +4G (site 10 in Table 5), as well as nucleotides downstream, on translation initiation has been inconclusive [46,49,50,51].”

What the sources say

• ?Not checked — no full text of the sourcecites [46] Kozak, 1999, Initiation of translation in prokaryotes and eukaryotes, Gene source
• ?Not checked — no full text of the sourcecites [49] Kozak, 2001, A progress report on translational control in eukaryotes, Sci. STKE source
• Disputed

  In the letter “Not every polymorphism close to the AUG codon can be explained by invoking context effects on initiation of translation,” Kozak says mutagenesis experiments found that +4G makes one of the strongest contributions to translation initiation. That is contrary to describing the effect of +4G as inconclusive. The References section also identifies a study reporting that +4G augments initiation, while nucleotides at +5 and +6 generally do not affect it. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  cites [50] Kozak, 2003, Not every polymorphism close to the AUG codon can be explained by invoking context effects on initiation of translation, Blood sourceExperimental — accuracy not yet measured
• Disputed

  The paper does not leave the effect of +4G inconclusive: the Discussion says its positive effect is generalizable, while the results report no significant effect from changes at +5 or +6 in the tests described. It notes a sequence-specific exception to the usual +4G effect, but presents that as an exception rather than an inconclusive overall finding (Discussion; Figures 4–6, page not provided in our copy). (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  cites [51] Kozak, 1997, Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6, Embo J. sourceExperimental — accuracy not yet measured

In the paper

“There is little evidence that +4G and downstream nucleotides contribute to translation initiation.”

What the sources say

• ?Not checked — no full text of the sourcecites [46] Kozak, 1999, Initiation of translation in prokaryotes and eukaryotes, Gene source
• ?Not checked — no full text of the sourcecites [49] Kozak, 2001, A progress report on translational control in eukaryotes, Sci. STKE source
• Disputed

  In the letter “Not every polymorphism close to the AUG codon can be explained by invoking context effects on initiation of translation” (page not supplied), Kozak says that G at +4 makes one of the strongest contributions to start-codon recognition. The paper also says downstream secondary structure can compensate for a suboptimal initiation context. Its caution about a particular −1 polymorphism does not support the broad claim that there is little evidence for a contribution from +4G. (LLM, conf=0.90)

  Checked by a reasoning model — may be wrong.

  cites [50] Kozak, 2003, Not every polymorphism close to the AUG codon can be explained by invoking context effects on initiation of translation, Blood sourceExperimental — accuracy not yet measured
• Partly supported

  The Discussion reports little or no effect from varying nucleotides at +5 and +6, supporting that part of the claim. But it also reports a strong, generalizable contribution of G at +4 to initiator-codon recognition, contrary to the claim about +4G. (LLM, conf=0.90)

  cites [51] Kozak, 1997, Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6, Embo J. sourceExperimental — accuracy not yet measured

In the paper

“Optimum codon usage without efficient translation initiation does not increase protein production [10,11] because translation initiation is often the rate-limiting step.”

What the sources say

• Disputed

  The Discussion and Conclusions (page unavailable) say that, for individual genes in the E. coli and S. cerevisiae transcriptomes studied, codon bias—not folding energy at the start of a gene—is the rate-limiting factor. The paper discusses effects on initiation, but its own conclusion does not support the citing sentence’s claim that initiation is often rate-limiting. (LLM, conf=0.83)

  Checked by a reasoning model — may be wrong.

  cites [10] Tuller, 2010, Translation efficiency is determined by both codon bias and folding energy, Proc. Natl. Acad. Sci. USA sourceExperimental — accuracy not yet measured
• Partly supported

  The Conceptual Framework says that inefficient initiation can limit the benefit of improved codon adaptation. But the Discussion concludes that both initiation and elongation limit translation efficiency, and notes that the low-initiation group studied is not representative of most genes. The paper therefore does not establish that initiation is often the rate-limiting step. (LLM, conf=0.83)

  cites [11] Xia, 2015, A Major Controversy in Codon-Anticodon Adaptation Resolved by a New Codon Usage Index, Genetics sourceExperimental — accuracy not yet measured

In the paper

“When nucleotide U in stop codons was replaced by Ψ, the rate of misreading of a stop codon by a near-cognate tRNAs increased [17].”

What the sources say

• Partly supported

  In “Targeted pseudouridylation promotes stop codon read-through” (page unavailable), the review reports that replacing U with Ψ promotes stop-codon read-through and describes tRNA anticodon pairing during decoding. It does not report a measured increase in the rate of misreading by near-cognate tRNAs specifically. (LLM, conf=0.82)

  cites [17] Adachi, 2019, Post-transcriptional pseudouridylation in mRNA as well as in some major types of noncoding RNAs, Biochim. Et Biophys. Acta. Gene Regul. Mech. sourceExperimental — accuracy not yet measured

In the paper

“Both mRNA encodes the same S-2P protein [5,6] which differ from the spike protein in the reference SARS-CoV-2 genome (NC_045512) by two amino acids, i.e., amino acids KV at sites 986 and 987 were replaced by PP to stabilize the resulting spike protein in the prefusion state to train the host immune system to recognize the virus before its entry into the host cell [7,8].”

What the sources say

• Partly supported

  The Methods section (“mRNA-1273 Vaccine”) states that mRNA-1273 encodes S-2P, a stabilized spike protein with two proline substitutions. The cited paper does not establish that the other mRNA referred to in the sentence encodes the same protein. (LLM, conf=0.86)

  cites [5] Anderson, E.J., Rouphael, N.G., Widge, A.T., Jackson, L.A., Roberts, P.C., Makhene, M., Chappell, J.D., Denison, M.R., Stevens, L.J., and Pruijssers, A.J. (2020). Safety and Immunogenicity of SARS-CoV-2 mRNA-1273 Vaccine in Older Adults. N. Engl. J. Med. sourceExperimental — accuracy not yet measured
• Partly supported

  The Methods > Vaccine passage states that mRNA-1273 encodes the S-2P antigen. The paper does not establish that both mRNA vaccines encode the same protein, so it supports only the mRNA-1273 part of the claim. (LLM, conf=0.87)

  cites [6] Jackson, 2020, An mRNA Vaccine against SARS-CoV-2-Preliminary Report, N. Engl. J. Med. sourceExperimental — accuracy not yet measured

In the paper

“Both mRNA encodes the same S-2P protein [5,6] which differ from the spike protein in the reference SARS-CoV-2 genome (NC_045512) by two amino acids, i.e., amino acids KV at sites 986 and 987 were replaced by PP to stabilize the resulting spike protein in the prefusion state to train the host immune system to recognize the virus before its entry into the host cell [7,8].”

What the sources say

• Partly supported

  The Methods section (‘mRNA-1273 Vaccine’; page not provided) says S-2P contains two proline substitutions, and the Discussion describes them as prefusion-stabilizing mutations. Our copy does not identify the original residues as KV, give positions 986 and 987, or specify the reference genome accession NC_045512. (LLM, conf=0.87)

  cites [5] Anderson, E.J., Rouphael, N.G., Widge, A.T., Jackson, L.A., Roberts, P.C., Makhene, M., Chappell, J.D., Denison, M.R., Stevens, L.J., and Pruijssers, A.J. (2020). Safety and Immunogenicity of SARS-CoV-2 mRNA-1273 Vaccine in Older Adults. N. Engl. J. Med. sourceExperimental — accuracy not yet measured
• Partly supported

  The Methods (Vaccine; page not provided) state that S-2P has two proline substitutions at positions 986 and 987, stabilizing the spike protein in its prefusion conformation. Our copy does not identify the original amino acids as KV or specify the reference genome accession NC_045512. (LLM, conf=0.84)

  cites [6] Jackson, 2020, An mRNA Vaccine against SARS-CoV-2-Preliminary Report, N. Engl. J. Med. sourceExperimental — accuracy not yet measured
• Partly supported

  The supplied passage describing S-2P (section labeled Abstract; page unavailable) says it contains two consecutive proline substitutions in the S2 subunit that stabilize the prefusion spike. Our copy does not identify the original residues as KV, give positions 986 and 987, or compare the construct with reference genome NC_045512, so those specifics are not verified. (LLM, conf=0.85)

  cites [7] Hsieh, 2020, Structure-based design of prefusion-stabilized SARS-CoV-2 spikes, Science sourceExperimental — accuracy not yet measured
• Supported

  Section 6, “The Spike Protein in Vaccine Development,” identifies S-2P as SARS-CoV-2 spike with the two proline substitutions K986P and V987P. It also says these substitutions stabilize the spike protein in its prefusion state. (LLM, conf=0.91)

  cites [8] Xia, X. (2021). Domains and Functions of Spike Protein in SARS-COV-2 in the Context of Vaccine Design. Viruses, 13. source

In the paper

“The codon optimization applied to BNT-162b2 and mRNA-1273 leads to a much increased codon adaptation index (CAI) [21,22] and index of translation efficiency (ITE) [11,36] for the two vaccine mRNAs.”

What the sources say

• Partly supported

  The 'Comparison of CAI with other indices' section defines CAI as a measure of synonymous codon usage bias, and 'Predicting levels of heterologous gene expression' discusses using it to assess whether a gene might benefit from more appropriate codons. The paper does not report CAI values for BNT-162b2 or mRNA-1273, so it cannot substantiate the claimed increase in those vaccine mRNAs. The ITE claim has separate citations and is not attributed to this source. (LLM, conf=0.88)

  cites [21] Sharp, 1987, The codon Adaptation Index--a measure of directional synonymous codon usage bias, and its potential applications, Nucleic Acids Res. sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [22] Xia, 2007, An Improved Implementation of Codon Adaptation Index, Evol. Bioinform. source
• Supported

  The citation is attached to the index of translation efficiency (ITE), rather than to the vaccine-specific increase. In “Necessity of a New Translation Elongation Index,” the paper defines ITE and explains how it is calculated. (LLM, conf=0.90)

  cites [11] Xia, 2015, A Major Controversy in Codon-Anticodon Adaptation Resolved by a New Codon Usage Index, Genetics source
• ?Not checked — no full text of the sourcecites [36] Xia, X. (2018). Bioinformatics and Translation Elongation. Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and Transcriptomics, Springer. source

In the paper

“Both mRNA encodes the same S-2P protein [5,6] which differ from the spike protein in the reference SARS-CoV-2 genome (NC_045512) by two amino acids, i.e., amino acids KV at sites 986 and 987 were replaced by PP to stabilize the resulting spike protein in the prefusion state to train the host immune system to recognize the virus before its entry into the host cell [7,8].”

What the sources say

• Supported

  The paper describes S-2P as a spike with two proline substitutions designed to stabilize the prefusion conformation. It also states that prefusion-stabilized glycoproteins are superior immunogens and that spike is a critical vaccine antigen and target of neutralizing antibodies because it mediates viral entry. Together, these passages support the stated vaccine rationale; the paper does not report a direct test of immune training by S-2P. (LLM, conf=0.86)

  cites [7] Hsieh, 2020, Structure-based design of prefusion-stabilized SARS-CoV-2 spikes, Science source
• Supported

  Section 6 states that the K986P and V987P substitutions stabilize the spike protein in its prefusion state. It also explains that presenting prefusion spike trains the immune system to act against the virus before the postfusion stage, when the virus is gaining entry into a cell. (LLM, conf=0.94)

  cites [8] Xia, X. (2021). Domains and Functions of Spike Protein in SARS-COV-2 in the Context of Vaccine Design. Viruses, 13. source

In the paper

“First, GC-rich mRNAs tend to be more stable than AU-rich mRNAs [32].”

What the sources say

• ?Could not verify

  The Results (Figure 4) report that silencing XRN1 stabilizes GC-rich mRNAs, while silencing PAT1B stabilizes AU-rich mRNAs. The Discussion describes distinct decay and storage pathways, but neither passage compares the groups’ overall stability. Searches for relative stability and half-life found no such comparison in our copy. (LLM, conf=0.74)

  cites [32] Courel, M., Clément, Y., Bossevain, C., Foretek, D., Vidal Cruchez, O., Yi, Z., Bénard, M., Benassy, M.N., Kress, M., and Vindry, C. (2019). GC content shapes mRNA storage and decay in human cells. eLife, 8. sourceExperimental — accuracy not yet measured

In the paper

“As mammalian host cells attack unmodified exogeneous RNA [12,13], all U nucleotides were replaced by N1-methylpseudouridine (Ψ) [14,15].”

What the sources say

• Supported

  The Introduction says unmodified RNA activates innate immune cells, while modified transcripts greatly diminish that response. The Results section reports that unmodified RNA induced inflammatory responses in human dendritic cells and mice; this supports the citing sentence’s informal description of host cells ‘attacking’ it. (LLM, conf=0.90)

  cites [12] Muramatsu, 2008, Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability, Mol. Ther. J. Am. Soc. Gene Ther. source
• ?Not checked — no full text of the sourcecites [13] Weissman, 2015, mRNA transcript therapy, Expert Rev. Vaccines source

In the paper

“Translation elongation becomes rate-limiting when translation initiation is highly efficient [10,11].”

What the sources say

• Partly supported

  The Discussion and Conclusions (page unavailable) says weak folding at the beginning of genes probably allows faster translation initiation and that, under this constraint in E. coli and S. cerevisiae, codon bias is rate-limiting for translation of individual genes. This supports the claim’s underlying link between initiation and elongation, but the paper does not establish the general rule that elongation becomes rate-limiting whenever initiation is highly efficient. (LLM, conf=0.82)

  cites [10] Tuller, 2010, Translation efficiency is determined by both codon bias and folding energy, Proc. Natl. Acad. Sci. USA sourceExperimental — accuracy not yet measured
• Supported

  The Conceptual Framework section explicitly states that elongation efficiency becomes rate-limiting when translation initiation efficiency is high. Its analysis also reports that protein production increases with elongation efficiency in the groups with more efficient initiation. (LLM, conf=0.94)

  cites [11] Xia, 2015, A Major Controversy in Codon-Anticodon Adaptation Resolved by a New Codon Usage Index, Genetics source

In the paper

“As mammalian host cells attack unmodified exogeneous RNA [12,13], all U nucleotides were replaced by N1-methylpseudouridine (Ψ) [14,15].”

What the sources say

• Not checked yetcites [14] European Medicines Agency (2021). Assessment report on Comirnaty (Common Name: COVID-19 mRNA Vaccine) (Nucleoside-Modified) Procedure No. EMEA/H/C/005735/0000, European Medicines Agency.
• Partly supported

  The Discussion (page not provided) says nucleoside modifications can reduce innate immune stimulation, and the Methods describe the vaccine mRNA. Neither passage specifies that every U nucleotide in mRNA-1273 was replaced with N1-methylpseudouridine. (LLM, conf=0.80)

  cites [15] Corbett, 2020, Evaluation of the mRNA-1273 Vaccine against SARS-CoV-2 in Nonhuman Primates, N. Engl. J. Med. sourceExperimental — accuracy not yet measured

In the paper

“This avoidance of CGN codons makes evolutionary sense given that the host zinc finger antiviral proteins (ZAP, gene name ZC3HAV1) target CpG dinucleotides in viral RNA and recruit cellular RNA degradation complexes to degrade the viral RNA genome [27,28,29].”

What the sources say

• Partly supported

  The paper’s main text (indexed under Abstract; no page given) reports that ZAP binds CG-enriched HIV-1 genome sequences and that direct ZAP recognition causes cytoplasmic depletion of high-CG RNA. It describes a screen involving RNA-degradation pathways, but does not show that ZAP recruits cellular RNA-degradation complexes to degrade the viral genome. (LLM, conf=0.86)

  cites [27] Takata, 2017, CG dinucleotide suppression enables antiviral defence targeting non-self RNA, Nature sourceExperimental — accuracy not yet measured
• Supported

  The Abstract identifies ZAP as ZC3HAV1 in mammals and says it binds specifically to CpG dinucleotides in viral RNA genomes. It also says ZAP recruits other proteins to degrade viral RNA genomes, supporting the attributed mechanism. (LLM, conf=0.95)

  cites [28] Xia, X. (2020). Extreme genomic CpG deficiency in SARS-CoV-2 and evasion of host antiviral defense. Mol. Biol. Evol., msaa094. source
• Partly supported

  The Introduction says ZAP targets CpG-rich viral RNA, and the Results section on ZAP isoforms says it promotes degradation of CpG-containing viral RNAs. Another Results section notes that ZAP lacks RNase activity and identifies cellular cofactors important for restriction, but our copy does not establish that ZAP recruits RNA-degradation complexes to degrade the viral genome. (LLM, conf=0.86)

  cites [29] Nchioua, R., Kmiec, D., Müller, J.A., Conzelmann, C., Groß, R., Swanson, C.M., Neil, S.J.D., Stenger, S., Sauter, D., and Münch, J. (2020). SARS-CoV-2 Is Restricted by Zinc Finger Antiviral Protein despite Preadaptation to the Low-CpG Environment in Humans. MBio, 11. sourceExperimental — accuracy not yet measured

In the paper

“Nearly half of human tRNA genes are not expressed [35]”

What the sources say

• Partly supported

  The Concluding Remarks say that approximately half of human tRNA genes are “silent or poorly expressed,” not that all of them are unexpressed. The discussion also notes that expression can vary by cell type and tissue, so the claim is more absolute than the source supports. (LLM, conf=0.86)

  cites [35] Torres, 2019, Enjoy the Silence: Nearly Half of Human tRNA Genes Are Silent, Bioinform. Biol. Insights sourceExperimental — accuracy not yet measured

In the paper

“In fact, multiple lines of evidence suggest much difference in termination efficiency and accuracy among the three stop codons.”

What the sources say

• ?Not checked — no full text of the sourcecites [63] Konecki, 1977, Characterization of reticulocyte release factor, J. Biol. Chem. source
• Partly supported

  The Results report that reticulocyte release factor recognizes all three stop codons and that a higher concentration of UAGA was needed for complete competition in a ribosome-binding assay. That is limited evidence of a difference in codon recognition, not a demonstration that termination efficiency or accuracy differs substantially among the three codons in eukaryotes. The paper also notes contamination of the UAGA preparation. (LLM, conf=0.78)

  cites [64] Tate, 1973, Influence of Guanine Nucleotides and Elongation Factors on Interaction of Release Factors with the Ribosome, Proc. Natl. Acad. Sci. USA sourceExperimental — accuracy not yet measured
• Partly supported

  The Results report some difference in termination efficiency among the three stop codons, but say it was much less pronounced than the effect of the base following the codon. The paper does not establish substantial differences in termination accuracy among the three codons; its experiments chiefly compare four-base stop signals in mammalian systems. (LLM, conf=0.87)

  cites [65] McCaughan, 1995, Translational termination efficiency in mammals is influenced by the base following the stop codon, Proc. Natl. Acad. Sci. USA sourceExperimental — accuracy not yet measured

In the paper

“Optimum codon usage without efficient translation initiation does not increase protein production [10,11] because translation initiation is often the rate-limiting step.”

What the sources say

• Partly supported

  The Results (page unavailable) find that mRNA folding modulates the association between codon bias and translation efficiency, with the strongest association when folding is very weak. That supports the idea that initiation-related folding can affect the benefit of codon usage, but the paper does not establish that optimal codon usage produces no increase in protein production when initiation is inefficient. The Discussion and Conclusions (page unavailable) instead identify codon bias as the rate-limiting factor for individual genes under the weak-folding conditions observed in the studied transcriptomes. (LLM, conf=0.85)

  cites [10] Tuller, 2010, Translation efficiency is determined by both codon bias and folding energy, Proc. Natl. Acad. Sci. USA sourceExperimental — accuracy not yet measured
• Supported

  The Conceptual Framework section says that only genes with efficient translation initiation are expected to increase protein production through improved codon-anticodon adaptation. It adds that low initiation efficiency implies little benefit from higher elongation efficiency, matching the cited claim. (LLM, conf=0.93)

  cites [11] Xia, 2015, A Major Controversy in Codon-Anticodon Adaptation Resolved by a New Codon Usage Index, Genetics source

In the paper

“Efficient translation initiation in mammalian species depends mainly on two factors [40,41]: (1) the Kozak consensus [42,43,44], and (2) the secondary structure that may embed the Kozak consensus to obscure these essential translation initiation signals [9,41,45].”

What the sources say

• ?Not checked — no full text of the sourcecites [40] Kozak, 1987, At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells, J. Mol. Biol. source
• ?Not checked — no full text of the sourcecites [41] Xia, X. (2018). Bioinformatics and Translation Initiation. Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and Transcriptomics, Springer. source
• ?Could not verify

  The Discussion section on pre-AUG poly(A) and Pab1p mentions secondary-structure stability in yeast and Drosophila IRESs, but not a Kozak consensus being obscured by secondary structure. The paper’s reported analysis concerns yeast poly(A) tracts; searches for Kozak and mammalian translation initiation found no passage addressing the attributed claim. Page numbers were not available in our copy. (LLM, conf=0.72)

  cites [9] Xia, 2011, Translation Initiation: A Regulatory Role for Poly(A) Tracts in Front of the AUG Codon in Saccharomyces cerevisiae, Genetics sourceExperimental — accuracy not yet measured
• Partly supported

  The study description reports that a very stable hairpin inhibits translation, supporting a role for secondary structure. But the Discussion says moderately stable hairpins often have no effect and argues against the proposed mechanism of hiding an upstream start codon. It also identifies the cap and sequences flanking the ATG codon as other regulators, so this paper does not establish that initiation depends mainly on the two stated factors or that secondary structure inhibits it by obscuring the Kozak consensus. (LLM, conf=0.88)

  cites [45] Kozak, 1986, Influences of mRNA secondary structure on initiation by eukaryotic ribosomes, Proc. Natl. Acad. Sci. USA sourceExperimental — accuracy not yet measured

In the paper

“This discontinuous mechanism of transcription [26,61] is confirmed by sequencing the subgenomic RNAs from cultured viruses (Figure 4C).”

What the sources say

• Supported

  The Introduction describes coronavirus transcription as producing subgenomic RNAs through paused negative-strand synthesis, with gene sequences joined to a common leader at transcription-regulatory sequences. That supports the cited description of a discontinuous mechanism. The sequencing confirmation is linked to the citing paper’s Figure 4C, rather than attributed to this source. (LLM, conf=0.88)

  cites [26] Alexandersen, 2020, SARS-CoV-2 genomic and subgenomic RNAs in diagnostic samples are not an indicator of active replication, Nat. Commun. source
• Supported

  The Results and Discussion state that sequencing reads spanning subgenomic RNA junctions confirm that SARS-CoV-2 uses canonical TRS-mediated template switching for discontinuous transcription. The Methods identify the sequenced RNA as coming from SARS-CoV-2-infected, cultured Vero cells. The confirmation concerns the canonical mechanism; the paper notes that some noncanonical transcripts may arise differently. (LLM, conf=0.92)

  cites [61] Kim, 2020, The Architecture of SARS-CoV-2 Transcriptome, Cell source

In the paper

“Efficient translation initiation in mammalian species depends mainly on two factors [40,41]: (1) the Kozak consensus [42,43,44], and (2) the secondary structure that may embed the Kozak consensus to obscure these essential translation initiation signals [9,41,45].”

What the sources say

• ?Not checked — no full text of the sourcecites [40] Kozak, 1987, At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells, J. Mol. Biol. source
• ?Not checked — no full text of the sourcecites [41] Xia, X. (2018). Bioinformatics and Translation Initiation. Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and Transcriptomics, Springer. source
• ?Not checked — no full text of the sourcecites [42] Kozak, 1980, Evaluation of the “scanning model” for initiation of protein synthesis in eucaryotes, Cell source
• Partly supported

  In the section on nucleotides flanking the initiator codon (page unavailable), Kozak proposes that favorable flanking nucleotides improve the efficiency with which ribosomes recognize an AUG. The paper examines eukaryotic messages and tests binding to wheat-germ ribosomes; it does not establish that mammalian translation initiation depends mainly on the consensus. It also says AUG position appears to be the primary determinant of a functional initiation site. (LLM, conf=0.84)

  cites [43] Kozak, 1981, Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes, Nucleic Acids Res. sourceExperimental — accuracy not yet measured
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites [44] Kozak, 1987, Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes, Mol. Cell. Biol.

In the paper

“Coronaviruses generate subgenomic mRNAs for translating structural proteins including the spike protein [59,60].”

What the sources say

• Partly supported

  The CORONAVIRUS REPLICATION AND TRANSCRIPTION section states that coronavirus transcription produces subgenomic mRNAs encoding structural proteins, and the REGULATION OF CORONAVIRUS PROTEIN STOICHIOMETRIC RATIOS section describes their translation into viral proteins. The passages do not identify spike specifically as one of those proteins, so that detail is not verified by our copy. (LLM, conf=0.82)

  cites [59] Sola, 2015, Continuous and Discontinuous RNA Synthesis in Coronaviruses, Annu. Rev. Virol. sourceExperimental — accuracy not yet measured
• Supported

  In “CoV RNA synthesis is confined to RO regions” (page unavailable), the paper says coronaviruses produce subgenomic RNAs encoding structural proteins and refers to these RNAs as subgenomic mRNAs. Its “Viral markers in the MERS-CoV RO” section identifies the S protein as a viral envelope protein, consistent with the spike-protein example. (LLM, conf=0.86)

  cites [60] Snijder, E.J., Limpens, R.W.A.L., de Wilde, A.H., de Jong, A.W.M., Zevenhoven-Dobbe, J.C., Maier, H.J., Faas, F.F.G.A., Koster, A.J., and Bárcena, M. (2020). A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis. PLOS Biol., 18. source

In the paper

“However, secondary structure downstream of the start codon tend to enhance recognition of start codons by eukaryotic ribosomes [57].”

What the sources say

• Supported

  The Discussion states that a downstream stem-loop increases recognition of an AUG codon in a suboptimal context. The Results add that the effect depends on the hairpin’s position, consistent with the claim’s qualified wording, “tend to enhance.” (LLM, conf=0.92)

  cites [57] Kozak, 1990, Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes, Proc. Natl. Acad. Sci. USA source

In the paper

“These studies suggest that UAA (especially UAAA) is more efficient than other stop signals.”

What the sources say

• ?Not checked — no full text of the sourcecites [63] Konecki, 1977, Characterization of reticulocyte release factor, J. Biol. Chem. source
• Partly supported

  The Results (Fig. 3; page unavailable) report that UAAA, UAGA, and UGAA all competed for release-factor binding, but that UAGA required a higher concentration for complete competition. This supports an advantage for UAAA over UAGA in that binding assay, not the broader claim that UAA or UAAA is more efficient than all other stop signals at termination. (LLM, conf=0.83)

  cites [64] Tate, 1973, Influence of Guanine Nucleotides and Elongation Factors on Interaction of Release Factors with the Ribosome, Proc. Natl. Acad. Sci. USA sourceExperimental — accuracy not yet measured
• Partly supported

  The Results report high termination activity for UAAA, using it as the reference for comparing the 12 signals. But they also report UGAA ≈ UAAA and say that the fourth base has a much stronger effect than the stop codon itself; they do not establish that UAA signals are generally more efficient than other stop signals. (LLM, conf=0.85)

  cites [65] McCaughan, 1995, Translational termination efficiency in mammals is influenced by the base following the stop codon, Proc. Natl. Acad. Sci. USA sourceExperimental — accuracy not yet measured

In the paper

“About 60% of the proteins experience N-terminal methionine excision (NME) which requires a small and nonpolar amino acid such as alanine and glycine.”

What the sources say

• ?Not checked — no full text of the sourcecites [52] Cigan, 1987, Sequence and structural features associated with translational initiator regions in yeast—A review, Gene source
• Partly supported

  The Introduction says initiator-methionine removal is most efficient when the second amino acid is small, and identifies alanine and glycine as the two smallest. But the Discussion says cleavage also occurs with valine at that position, so the claim overstates this preference as a requirement. (LLM, conf=0.85)

  cites [53] Xia, X. (2007). The +4G site in Kozak consensus is not related to the efficiency of translation initiation. PLoS ONE, 2. sourceExperimental — accuracy not yet measured

In the paper

“About 60% of the proteins experience N-terminal methionine excision (NME) which requires a small and nonpolar amino acid such as alanine and glycine.”

What the sources say

• ?Not checked — no full text of the sourcecites [52] Cigan, 1987, Sequence and structural features associated with translational initiator regions in yeast—A review, Gene source
• Partly supported

  The Introduction (page not available in our copy) discusses removal of the initiator methionine and says cleavage is most efficient when the second amino acid is small, such as alanine or glycine. It gives no estimate that about 60% of proteins undergo this removal; its statement that amino-terminal modifications occur in nearly all proteins does not establish the frequency of methionine excision specifically. (LLM, conf=0.86)

  cites [53] Xia, X. (2007). The +4G site in Kozak consensus is not related to the efficiency of translation initiation. PLoS ONE, 2. sourceExperimental — accuracy not yet measured

In the paper

“As demonstrated in previous studies on translation initiation in yeast [55] and in mouse cell lines [56], stable secondary structure in the 5′-UTR before or flanking the start codon decreases protein production.”

What the sources say

• Partly supported

  The Results (Fig. 3; page unavailable) report that more stable structures near the start codon are associated with reduced protein levels in yeast. But the paper describes this as a correlation and says such structures may impede ribosomal scanning; it does not demonstrate that the structures themselves cause the decrease asserted in the citing sentence. (LLM, conf=0.85)

  cites [55] Dvir, 2013, Deciphering the rules by which 5′-UTR sequences affect protein expression in yeast, Proc. Natl. Acad. Sci. USA sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [56] Pelletier, 1985, Insertion mutagenesis to increase secondary structure within the 5′ noncoding region of a eukaryotic mRNA reduces translational efficiency, Cell source

In the paper

“The associated position weight matrix [23,24,47,48], using intron nucleotide frequencies (0.26398, 0.21777, 0.22622 and 0.29203 for A, C, G, and T, respectively) as background frequencies, shows a site-specific pattern consistent with the GCCRCCAUGG consensus.”

What the sources say

• Partly supported

  The Introduction (page unavailable) describes position weight matrices as tools for characterizing nucleotide motifs, including translation initiation sites. Section 2.1 discusses choosing background nucleotide frequencies for a PWM. The paper does not report a PWM for human chromosome 1 start-codon flanks, the stated intron frequencies, or a pattern matching GCCRCCAUGG. (LLM, conf=0.84)

  cites [23] Xia, 2012, Position Weight Matrix, Gibbs Sampler, and the Associated Significance Tests in Motif Characterization and Prediction, Scientifica sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [24] Xia, X. (2018). Position weight matrix and Perceptron. Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and Transcriptomics, Springer. source
• ?Not checked — no full text of the sourcecites [47] Hertz, 1990, Identification of consensus patterns in unaligned DNA sequences known to be functionally related, Comput. Appl. Biosci. source
• Partly supported

  The Abstract describes using matrices to evaluate nucleotide sequences, supporting the citation’s connection to a matrix-based method. It does not report a position weight matrix for human chromosome 1 genes, the stated intron background frequencies, or a pattern matching GCCRCCAUGG; those empirical details are not supported by our copy. (LLM, conf=0.82)

  cites [48] Stormo, 1986, Quantitative analysis of the relationship between nucleotide sequence and functional activity, Nucleic Acids Res. sourceExperimental — accuracy not yet measured

In the paper

“G/Ψ base pairs are expected to be stronger than G/U base pairs [16].”

What the sources say

• Supported

  The Discussion reports that replacing U with Ψ in Ψ-G pairs generally enhances duplex stability: across six duplexes, the enhancement relative to U-G ranges from 0 to 1.4 kcal/mol. The paper notes that the effect depends on neighboring bases, consistent with the claim's phrasing as an expectation rather than an absolute rule. (LLM, conf=0.92)

  cites [16] Kierzek, 2014, The contribution of pseudouridine to stabilities and structure of RNAs, Nucleic Acids Res. source

In the paper

“First, while the -3R (site 4 in the first column of Table 5) has been demonstrated repeatedly to enhance translation initiation, the effect of +4G (site 10 in Table 5), as well as nucleotides downstream, on translation initiation has been inconclusive [46,49,50,51].”

What the sources say

• ?Not checked — no full text of the sourcecites [46] Kozak, 1999, Initiation of translation in prokaryotes and eukaryotes, Gene source
• ?Not checked — no full text of the sourcecites [49] Kozak, 2001, A progress report on translational control in eukaryotes, Sci. STKE source
• Supported

  In the main text (page not provided), Kozak states that mutagenesis experiments showed A or G at position −3 makes one of the strongest contributions to start-codon recognition. The paper also says its context rules were established in cultured cells and replicated in vitro, supporting the claim that this effect has been demonstrated repeatedly. (LLM, conf=0.91)

  cites [50] Kozak, 2003, Not every polymorphism close to the AUG codon can be explained by invoking context effects on initiation of translation, Blood source
• Supported

  The paper describes A at position −3 as optimal and reports that mutating A−3 nearly abolished initiation from CUG or ACG codons (text preceding the Discussion; page unavailable in our copy). This supports the attributed point that a purine at −3 enhances translation initiation. (LLM, conf=0.88)

  cites [51] Kozak, 1997, Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6, Embo J. source

In the paper

“In mammalian genes, the effect of the +4 site is consistent among all three different stop codons in experimental studies, with termination efficiency of UAAR >> UAAY, UAGR >> UAGY and UGAR >> UGAY both in vitro and in vivo [65].”

What the sources say

• Partly supported

  The abstract and Discussion report that a +4 purine strengthens termination for all three stop codons in vitro. The Results report the corresponding in vivo comparison only for UGA, not for UAA and UAG, so the claim overextends the in vivo finding. (LLM, conf=0.89)

  cites [65] McCaughan, 1995, Translational termination efficiency in mammals is influenced by the base following the stop codon, Proc. Natl. Acad. Sci. USA sourceExperimental — accuracy not yet measured

In the paper

“Based on the observed contact between amino acids in a protein and bases in DNA, glycine interacts far more favorably with purine than with pyrimidine [92].”

What the sources say

• ?Could not verify

  The Hydrogen bonds section reports that glycine makes some interactions, and the van der Waals contacts section says its overall contact frequency is as expected. Neither section compares glycine’s interactions with purines and pyrimidines; searches for glycine with the individual bases and for a purine preference found no such comparison in our copy. (LLM, conf=0.73)

  cites [92] Luscombe, 2001, Amino acid-base interactions: A three-dimensional analysis of protein-DNA interactions at an atomic level, Nucleic Acids Res. sourceExperimental — accuracy not yet measured

In the paper

“Highly expressed genes in diverse bacterial species strongly prefer UAA codons even in strongly GC-rich genomes with an overwhelming majority of stop codons being UGA [74,75].”

What the sources say

• Supported

  The Results and Discussion section reports that UAA is more frequent in highly expressed genes than in lowly expressed genes across all 14 species, including extremely GC-biased genomes. It also reports that UGA accounts for an overwhelming majority of stop codons in P. aeruginosa, where highly expressed genes nevertheless favor UAA relative to lowly expressed genes. Page numbers are not provided in our copy. (LLM, conf=0.94)

  cites [74] Wei, 2016, Coevolution between Stop Codon Usage and Release Factors in Bacterial Species, Mol. Biol. Evol. source
• ?Not checked — no full text of the sourcecites [75] Xia, X. (2018). Bioinformatics and Translation Termination in Bacteria. Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and Transcriptomics, Springer. source

In the paper

“For example, the termination efficiency of UGAC is <20% of UAAA [65].”

What the sources say

• Supported

  The Results report that UGAC is the weakest and UGAA the strongest signal in the UGAN series, which spans an approximately eightfold range in the in vitro termination assay. They also report UGAA ≈ UAAA, supporting the claim that UGAC's efficiency is less than 20% of UAAA's in that assay. Page numbers are unavailable in our copy. (LLM, conf=0.86)

  cites [65] McCaughan, 1995, Translational termination efficiency in mammals is influenced by the base following the stop codon, Proc. Natl. Acad. Sci. USA source

In the paper

“To alleviate host cells to attack exogeneous vaccine mRNA as foreign RNA [13,58], all uridines in the mRNA vaccines were replaced by Ψ [2,14].”

What the sources say

• Supported

  The Methods section (Pre-clinical mRNA-1273 mRNA and LNP Production Process) states that the vaccine mRNA was synthesized with complete replacement of uridine by N1-methylpseudouridine. This confirms the claim for mRNA-1273, the vaccine examined in this source. (LLM, conf=0.93)

  cites [2] Corbett, 2020, SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness, Nature source
• Not checked yetcites [14] European Medicines Agency (2021). Assessment report on Comirnaty (Common Name: COVID-19 mRNA Vaccine) (Nucleoside-Modified) Procedure No. EMEA/H/C/005735/0000, European Medicines Agency.

In the paper

“Second, highly expressed genes in the yeast prefer UAA stop codons [76].”

What the sources say

• Partly supported

  The Background and Discussion report that stop codon usage correlates with transcription level in yeast, but do not identify UAA as the codon preferred by highly expressed yeast genes. The Results establish a UAA preference in highly expressed human genes, so the claim extends that specific finding to yeast without support from this paper. (LLM, conf=0.87)

  cites [76] Trotta, E. (2016). Selective forces and mutational biases drive stop codon usage in the human genome: A comparison with sense codon usage. BMC Genom., 17. sourceExperimental — accuracy not yet measured

In the paper

“Stop codon UGA with a +4C is most prone to readthrough in mammalian genes, especially in the context of UGA CUA [77,78].”

What the sources say

• Supported

  The Introduction identifies UGA CUA (G) as a consensus context for high translational readthrough in mammals. The Results report that changing the +4 C in the UGA CUA context reduces readthrough of human MDH1 to background levels, supporting the emphasis on that base and sequence. (LLM, conf=0.90)

  cites [77] Hofhuis, J., Schueren, F., Nötzel, C., Lingner, T., Gärtner, J., Jahn, O., and Thoms, S. (2016). The functional readthrough extension of malate dehydrogenase reveals a modification of the genetic code. Open Biol., 6. source
• Supported

  The Introduction says UGA is the stop codon most likely to allow readthrough in humans. In Results (‘A consensus for high readthrough in humans’), the paper identifies UGA CUA—which has C at position +4—as a consensus for high basal readthrough. (LLM, conf=0.91)

  cites [78] Schueren, 2014, Peroxisomal lactate dehydrogenase is generated by translational readthrough in mammals, eLife source

In the paper

“However, in many cases, misreading UGA stop codons in prokaryotes is associated with a +1 frameshift [81,82,83,84].”

What the sources say

• ?Not checked — no full text of the sourcecites [81] Craigen, 1986, Expression of peptide chain release factor 2 requires high-efficiency frameshift, Nature source
• Partly supported

  The paper’s Discussion proposes a frameshift near an in-frame UGA codon in E. coli release factor 2, and describes a tRNA reported to translocate four bases, consistent with a +1 shift. It does not establish that UGA misreading is associated with +1 frameshifts in many prokaryotic cases; its discussion of other frameshifting examples does not make that broader claim. (LLM, conf=0.86)

  cites [82] Craigen, 1985, Bacterial peptide chain release factors: Conserved primary structure and possible frameshift regulation of release factor 2, Proc. Natl. Acad. Sci. USA sourceExperimental — accuracy not yet measured
• Partly supported

  The Introduction describes a +1 frameshift at the CUU–UGA site in the E. coli RF2 gene, and the Discussion says an adjacent stop codon can increase frameshifting. The paper describes a tRNA shifting at the codon before UGA, not misreading UGA itself, and it does not establish that this occurs in many cases across prokaryotes. (LLM, conf=0.86)

  cites [83] Weiss, 1988, Reading frame switch caused by base-pair formation between the 3′ end of 16S rRNA and the mRNA during elongation of protein synthesis in Escherichia coli, Embo J. sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [84] Curran, 1988, Use of tRNA suppressors to probe regulation of Escherichia coli release factor 2, J. Mol. Biol. source

In the paper

“The first involves the codon usage of human ribosomal protein genes (“RP” in Table 1) which are known to be highly expressed. These genes prefer CGC codons (Table 1).”

In the paper

“almost all UUR codons for Leu were recoded to CUN codons in both vaccine mRNAs (Table 1).”

In the paper

“Both codon subfamilies are used roughly equally for encoding Ser.”

In the paper

“In contrast, Pfizer/BioNTech’s BNT-162b2 took the liberal strategy, and left 14 GAA codons unchanged (Table 3).”

In the paper

“However, human genes use CGN more frequently than AGR codons for encoding Arg.”

In the paper

“In contrast, the UCN subfamily has both UCC and UCU used frequently.”

In the paper

“Among the ribosomal protein genes (33 RPS and 50 RPL) known to be highly expressed, 64.2% of the Arg residues are encoded by CGN codons.”

In the paper

“This is not a concern with the intramuscular injection because, according to three sets of gene expression data from Human Protein Atlas at http://www.proteinatlas.org (accessed on 20 June 2021) [30], ZAP is almost absent in skeletal muscle (Figure 1).”

What the sources say

• ?Not checked — no full text of the sourcecites [30] Fagerberg, 2015, Proteomics. Tissue-based map of the human proteome, Science source

In the paper

“Consequently, G-ending codons were taken as the optimal codon in the two mRNA vaccines (Table 3 for GAR codons encoding Glu).”

In the paper

“Similarly, the 3′-UTR of this sequence is identical to one of the 3′-UTR sequences (SEQ ID NO 21) in another Moderna patent [19], except that the seventh triplet is UAG in the putative sequence instead of AUG.”

What the sources say

• Not checked yetcites [19] De Fougerolles, A., and Guild, J. (2020). Modified Polynucleotides for the Production of Secreted Proteins. (US 10,703,789 B2), U.S. Patent.

In the paper

“Highly expressed human ribosomal protein genes encode 81% of Leu by CUN codons.”

In the paper

“Note that mutation bias would have favored U-ending codons because the frequency of U in introns is higher than that of C (0.2920 for U and 0.2178 for C, Table 1)”

In the paper

“but highly expressed ribosome protein genes prefer AGC over AGU (144 for AGC and 95 for AGU, Table 1).”

In the paper

“The remaining 26 genes (Table 2), including the most muscle-specific genes such as titin (TTN), actin (ACTA1) and myosin (MYH1), use CGC codons significantly more than CGG codons (Paired sample t-test, t = 3.075, DF = 25, p = 0.0034, 2-tailed test).”

In the paper

“Its values are similarly much higher in the two vaccine mRNAs than in natural viruses.”

In the paper

“Second, CGG seems to have more tRNA decoding it than other synonymous CGN codons.”

In the paper

“They also use more CGC than CGG (CGC:CGG are 3:0 for hEF1A1, 8:6 for hEF1A2, and 14:4 for hPABPC1).”

In the paper

“The two vaccines recoded nearly all CGN codons to CGG (Table 1).”

In the paper

“However, the 5′-UTR of this putative sequence is identical to one of the 5′-UTR sequences (SEQ ID NO 181) in a Moderna patent [18].”

What the sources say

• Not checked yetcites [18] Huang, E.Y.-C., Tse, S.-W., Iacovelli, J., McKinney, K., and Valiante, N. (2021). Immunomodulatory Therapeutic mRNA Compositions Encoding Activating Oncogene Mutation Peptides. (US 10,881,730 B2), U.S. Patent.

In the paper

“For this reason, many Ser codons UCN were recoded to AGC in the vaccine mRNAs, especially in mRNA-1273 (Table 1).”

In the paper

“Moderna’s mRNA-1273 has taken the fundamentalist strategy and replaced all GAA codons by GAG.”

In the paper

“The 5′-UTR from naturally produced spike mRNA was derived from the analysis.”

In the paper

“The subgenomic SARS-2-S mRNA, recovered from sequencing, does not include the first 25 nt in the leader sequence.”

In the paper

“The MFE for the 40 nucleotides with the start codon in the middle is -12.3 for mRNA-1273 at 37 °C.”

In the paper

“However, Moderna’s mRNA-1273 is peculiar in having a secondary structure flanking the start codon (Figure 2B) that is visualized in Figure 2C.”

In the paper

“For example, 8.6% of ribosomal protein genes (including different isoforms) have MFE values for the 40 nt windowd equal or smaller than −12.3 (Figure 3).”

In the paper

“The 5′-UTR in Moderna’s mRNA-1273 is V1-UTR (SEQ ID NO 181) [18].”

What the sources say

• Not checked yetcites [18] Huang, E.Y.-C., Tse, S.-W., Iacovelli, J., McKinney, K., and Valiante, N. (2021). Immunomodulatory Therapeutic mRNA Compositions Encoding Activating Oncogene Mutation Peptides. (US 10,881,730 B2), U.S. Patent.

In the paper

“Moderna has developed many alternative 5′-UTRs and 3′-UTR sequences listed in two patents [18,19].”

What the sources say

• Not checked yetcites [18] Huang, E.Y.-C., Tse, S.-W., Iacovelli, J., McKinney, K., and Valiante, N. (2021). Immunomodulatory Therapeutic mRNA Compositions Encoding Activating Oncogene Mutation Peptides. (US 10,881,730 B2), U.S. Patent.
• Not checked yetcites [19] De Fougerolles, A., and Guild, J. (2020). Modified Polynucleotides for the Production of Secreted Proteins. (US 10,703,789 B2), U.S. Patent.

In the paper

“BNT162b2 has little secondary structure flanking the start codon that is located at mid-window sites 55–57 (Figure 2B).”

In the paper

“The design of the 5′-UTR of BNT162b2 took the first approach by incorporating the 5′-UTR of human α-globin (5′-UTR is identical between human HBA1 and HBA2) with a minor modification of the Kozak consensus (Figure 2A).”

In the paper

“For example, among 11,327 annotated protein-coding genes and their splice isoforms in human chromosome 1 (Chr01, NC_000001.11), only five genes (OAZ3, FNDC5, FNDC5, RNF187 and WDR26) have one of its isoforms featuring a non-AUG start codon.”

In the paper

“The long stem in Figure 2 has a G/U base pair to close the stem.”

In the paper

“Second, the second amino acid in the spike protein is phenylalanine, which ensures that NME does not happen.”

In the paper

“Empirical data in Table 6 shows that highly expressed mammalian genes also prefer UAA stop codons.”

In the paper

“The functionally important and highly expressed RP genes use +4R much more than an average gene represented by Chr01 genes.”

In the paper

“The difference, when tested by a likelihood ratio test, is highly significant (likelihood ratio chi-square = 931.0514, DF = 17, p < 0.000001).”

In the paper

“For this reason, codon preference by highly expressed genes relative to lowly expressed genes is a better operational criterion for codon optimization.”

In the paper

“For example, GAΨ encoding Asp could pair with the anticodon of tRNAGlu leading to nonsynonymous substitutions.”

In the paper

“This is likely to increase misreading of a codon by a near-cognate tRNA.”

In the paper

“The near absence of ZAP in muscle cells suggests that spike mRNAs in the vaccine injected into muscle cells will not be subject to ZAP-mediated RNA degradation.”

In the paper

“First, the EMBOSS [33] compilation of codon usage, which is frequently used in codon optimization, shows that CGG is used slightly more frequently than CGC.”

What the sources say

• ?Not checked — no full text of the sourcecites [33] Rice, 2000, EMBOSS: The European Molecular Biology Open Software Suite, Trends Genet. source

In the paper

“This avoidance of CGN codons makes evolutionary sense given that the host zinc finger antiviral proteins (ZAP, gene name ZC3HAV1) target CpG dinucleotides in viral RNA and recruit cellular RNA degradation complexes to degrade the viral RNA genome [27,28,29].”

In the paper

“The smaller value of CAI and ITE values for BNT-162b2 than mRNA-1273 might give an impression that BNT-162b2 is less codon-optimized than mRNA-1273. This is not necessarily true.”

In the paper

“In particular, I point out a few optimizations in the design of the two mRNA vaccines that have not been performed properly.”

• Also stated in: § 1. Introduction · plain

  “By using genomic and transcriptomic data, I pointed out a few inappropriately performed optimizations in the design of the two mRNA vaccines.”

In the paper

“This would imply that mRNA in the Pfizer/BioNTech vaccine on average likely produces about 3.3 times as many proteins as an mRNA in the Moderna vaccine.”

In the paper

“The designers of the mRNA vaccines (especially mRNA-1273, Table 1) chose a wrong codon as the optimal codon.”

In the paper

“These multiple lines of evidence suggest that CGC is a better codon than CGG.”

In the paper

“The fundamentalist strategy will necessarily generate higher CAI or ITE values than the liberal strategy.”

In the paper

“This is not a concern with the intramuscular injection because, according to three sets of gene expression data from Human Protein Atlas at http://www.proteinatlas.org (accessed on 20 June 2021) [30], ZAP is almost absent in skeletal muscle (Figure 1).”

In the paper

“so the slightly higher usage of CGG than CGC may simply be due to mutation bias”

In the paper

“Therefore, the CGC codon preferred by ribosomal protein genes are also preferred by highly expressed muscle genes.”

In the paper

“Second, in the unlikely case when the vaccine mRNAs were recombined into a SARS-CoV-2 virus, the result would not be a virus with an optimized spike protein gene, but a segment of CpG-rich RNA that would be targeted by host ZAP for degradation.”

In the paper

“so we cannot use tRNA gene copy number as a proxy of tRNA abundance in the cellular tRNA pool.”

In the paper

“The sequence of mRNA-1273 was taken from Jeong et al. [4], and should be considered putative.”

What the sources say

• Not checked yetcites [4] Jeong, D.-E., McCoy, M., Artiles, K., Ilbay, O., Fire, A., Nadeau, K., Park, H., Betts, B., Boyd, S., and Hoh, R. (2021, June 20). Assemblies-of-Putative-SARS-CoV2-Spike-Encoding-mRNA-Sequences-for-Vaccines-BNT-162b2-and-mRNA-1273. Available online: https://virological.org/t/assemblies-of-putative-sars-cov2-spike-encoding-mrna-sequences-for-vaccines-bnt-162b2-and-mrna-1273/663.

In the paper

“The transcription of the subgenomic negative strand pauses at TRS-B, resulting in the negative strand shifting position from base-pairing with TRS-B to base-pairing with TRS-L (Figure 4B).”

In the paper

“The codon optimization in the previous section suffers from the lack of consideration for translation accuracy [36,37].”

What the sources say

• ?Not checked — no full text of the sourcecites [36] Xia, X. (2018). Bioinformatics and Translation Elongation. Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and Transcriptomics, Springer. source
• ?Not checked — no full text of the sourcecites [37] Akashi, 1994, Synonymous codon usage in Drosophila melanogaster: Natural selection and translational accuracy, Genetics source

In the paper

“He found greater codon adaptation in conserved amino acid sites than in variable amino acid sites.”

What the sources say

• ?Not checked — no full text of the sourcecites [37] Akashi, 1994, Synonymous codon usage in Drosophila melanogaster: Natural selection and translational accuracy, Genetics source

In the paper

“In other words, the secondary structure of these 8.6% of ribosomal protein genes are equally stable or even more stable than that of mRNA-1723.”

In the paper

“Second, the second amino acid in the spike protein is phenylalanine, which ensures that NME does not happen.”

In the paper

“The Kozak consensus for mammalian genes that enhances translation initiation is GCCRCCAUGG [40,46], where AUG is the start codon.”

What the sources say

• ?Not checked — no full text of the sourcecites [40] Kozak, 1987, At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells, J. Mol. Biol. source
• ?Not checked — no full text of the sourcecites [46] Kozak, 1999, Initiation of translation in prokaryotes and eukaryotes, Gene source

In the paper

“This GC-rich element, and the secondary structure it contributes to (Figure 2C) may increase mRNA stability and translation accuracy by reducing leaky scanning [18].”

What the sources say

• Not checked yetcites [18] Huang, E.Y.-C., Tse, S.-W., Iacovelli, J., McKinney, K., and Valiante, N. (2021). Immunomodulatory Therapeutic mRNA Compositions Encoding Activating Oncogene Mutation Peptides. (US 10,881,730 B2), U.S. Patent.

In the paper

“These factors contribute to the efficiency of ribosomes being properly positioned at start codon to transit from translation initiation to elongation.”

In the paper

“The coding sequence (CDS) of the spike protein, as well as all downstream coding sequences, features a TRS (TRS-B) upstream of the CDS.”

In the paper

“However, the 5′-UTR of BNT162b2 derived from 5′-UTR of the human α-globin gene (Figure 2) does appear to be superior over mRNA-1273 in this aspect.”

In the paper

“This leader sequence includes a transcription regulatory sequence (TRS-L) at its 3′ end (Figure 4A).”

In the paper

“Changing it to GCG (encoding alanine) would result in NME leading to unpredictable changes in the S protein.”

In the paper

“Therefore, the secondary structure in mRNA-1273 (Figure 2) does not necessarily imply low translation efficiency.”

In the paper

“All subgenomic mRNA transcripts share the same 5′ end leader sequence located at the 5′ end of the genomic sequence (sites 1–70, Figure 4A).”

In the paper

“However, AAC and AAU also differ in misreading rate, in particular by tRNALys, which ideally should decode only AAA and AAG codons but does misread AAC and AAU, leading to Asn replaced by Lys.”

What the sources say

• ?Not checked — no full text of the sourcecites [38] Johnston, 1984, Codon specificity of starvation induced misreading, Mol. Gen. Genet. source
• ?Not checked — no full text of the sourcecites [39] Johnston, 1985, Streptomycin-induced, third-position misreading of the genetic code, J. Mol. Biol. source

In the paper

“Subgenomic mRNAs for the spike protein gene and all other ORFs downstream share the same leader sequence and the 3′-UTR.”

In the paper

“I disregard the nature of start codon as a relevant factor contributing to translation initiation efficiency because there is little variation in start codon usage in mammalian genes.”

In the paper

“However, there are several inappropriate optimizations.”

In the paper

“The readthrough of UAA occurs at frequencies from 9 × 10−4 to less than 1 × 10−5 [70].”

What the sources say

• ?Not checked — no full text of the sourcecites [70] Ryden, 1984, A temperature-sensitive mutant of Escherichia coli that shows enhanced misreading of UAG/A and increased efficiency for some tRNA nonsense suppressors, Mol. Gen. Genet. source