Modifications of mRNA vaccine structural elements for improving mRNA stability and translation efficiency

A higher GC sequence translates 100-fold higher than a low GC sequence.

“Although high GC-content may cause problems for mRNA secondary structure, the higher GC sequence translates 100-fold higher than a low GC sequence (Kudla et al. 2006).”

• cites Kudla et al. 2006 sourceDisputed

  The Results (page unavailable) attribute higher protein output from GC-rich GFP to higher mRNA levels, not a higher translation rate. The Discussion likewise says codon optimization increased GFP mRNA levels rather than its translation rate. The claimed 100-fold increase in translation is therefore contrary to the paper's finding. (LLM, conf=0.90)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

α- and β-globin 3′ UTRs contain sequence features that enhance the translation and stability of mRNA.

“The 3ʹ-UTRs of α- and β-globin mRNAs are found in many in vitro-transcribed (IVT) mRNA, and they contain sequence features that enhance the translation and stability of mRNA (Sahin et al. 2014; Karikó et al. 1999).”

• cites Sahin et al. 2014 sourceSupported
• cites Karikó et al. 1999 sourceNot checked — no full text of the source

Modified nucleosides can be used to create mRNA vaccines with significantly improved translation efficiency.

“The modified nucleosides escape the type interferons (IFNs) induced antiviral pathways and are targeted to break and block invading mRNA; therefore they can be used to create mRNA vaccines with significantly improved translation efficiency (Verbeke et al. 2019).”

• cites Verbeke et al. 2019 sourceSupported

The translation elongation rate is controlled by the accessibility of cognate tRNA species and codon usage modification.

“The translation elongation rate is controlled by the accessibility of cognate tRNA species and the codon usage modification to prevent the match rare tRNA sequence of species and integrate sequences that match more common tRNA species (Hanson and Coller 2018).”

• cites Hanson and Coller 2018 sourceSupported

5′-capping promotes pre-mRNA splicing.

“Screening mRNA against exonuclease activity, promoting pre-mRNA splicing, and functioning as binding site of eIF4F heterodimeric translation initiation complex are all important functions of 5′-capping (Izaurralde et al. 1994; Gallie 1991).”

• cites Izaurralde et al. 1994 sourceNot checked — no full text of the source
• cites Gallie 1991 sourceCould not verify

  The paper’s opening discussion and Discussion section address the cap’s roles in translation and mRNA stability, not pre-mRNA splicing. Searches for splicing and for cap-dependent removal of introns found no passage stating that capping promotes splicing; a splicing study appears only in the References. (LLM, conf=0.75)

  Experimental — accuracy not yet measured

The 5′-cap structure is a key factor in the host's ability to differentiate between self and non-self mRNA molecules.

“The 5′-cap structure is a key factor in the host's ability to differentiate between self and non-self mRNA molecules (Devarkar et al. 2016).”

• cites Devarkar et al. 2016 sourceSupported

The structural characteristics of mRNA such as the CAP, poly(A) tail, and UTRs directly impact mRNA stability and translation efficiency.

“The structural characteristics of mRNA such as the CAP, poly(A) tail, and untranslated regions (UTRs) directly impact mRNA stability and translation efficiency (Orlandini von Niessen et al. 2019; Holtkamp et al. 2006).”

• cites Orlandini von Niessen et al. 2019 sourceNot checked — no full text of the source
• cites Holtkamp et al. 2006 sourceSupported

Shorter 5′UTRs are better for mRNA translation.

“(iii) Shorter 5′UTRs may be used, as past research has indicated that this type of 5′UTR is better for mRNA translation (Leppek et al. 2018).”

• cites Leppek et al. 2018 sourceCould not verify

  The section ‘5′ UTRs as platforms for RNA structure’ describes variation in UTR length but does not compare translation efficiency by length. ‘5′ UTR structures in ribosome scanning’ notes that some exceptionally short UTRs permit scanning-free initiation; it does not say shorter UTRs are generally better for translation. Searches for short UTRs and translational efficiency found no such comparison in our copy. (LLM, conf=0.70)

  Experimental — accuracy not yet measured

The 3′ UTRs of α- and β-globin mRNAs are found in many in vitro-transcribed (IVT) mRNA.

“The 3ʹ-UTRs of α- and β-globin mRNAs are found in many in vitro-transcribed (IVT) mRNA, and they contain sequence features that enhance the translation and stability of mRNA (Sahin et al. 2014; Karikó et al. 1999).”

• cites Sahin et al. 2014 sourceSupported
• cites Karikó et al. 1999 sourceNot checked — no full text of the source

The steady effect of human β-globin 3′ UTR sequences is enhanced when two human β-globin 3′ UTRs are placed in a head-to-tail orientation.

“The steady effect of human β-globin 3ʹ-UTR sequences is enhanced when two human β-globin 3ʹ-UTRs are placed in a head-to-tail orientation (Holtkamp et al. 2006).”

• cites Holtkamp et al. 2006 sourceSupported

The length of the poly(A) tail is proportional to translation efficiency.

“The poly(A) tail steadies mRNA and boosts protein translation, and the length of the poly(A) tail is proportional to translation efficiency.”

• cites Park et al. 2016 sourcePartly supported

  In “Translational Regulation and Poly(A) Tail Control during the Somatic Cell Cycle” (Figure 3B), the paper reports a relationship between tail length and translation efficiency only for short tails, up to about 20 nt. It says lengthening beyond that does not further influence translation, so the unqualified claim of proportionality overstates the finding. (LLM, conf=0.90)

  Experimental — accuracy not yet measured
• cites Jalkanen et al. 2014 sourcePartly supported

  The Introduction says poly(A) tails can enhance translation efficiency. But “Does size matter?” reports a relationship between tail length and translation in early development and certain other contexts, while noting that a global analysis found no significant correlation beyond embryonic stages. The source therefore does not support an unqualified, proportional relationship. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

mRNA secondary structure design can be modified to improve mRNA stability against endonuclease cleavage and chemical degradation.

“Furthermore, mRNA secondary structure design can be modified to improve mRNA stability against endonuclease cleavage and chemical degradation (Wayment-Steele et al. 2021).”

• cites Wayment-Steele et al. 2021 sourceSupported

Codon modification is critical for mRNA stability.

“Furthermore, the rate of codon-dependent translation elongation has been a primary factor of mRNA stability, codon modification is critical for mRNA stability (Presnyak et al. 2015).”

• cites Presnyak et al. 2015 sourceSupported

The poly(A) tail is a critical factor in determining the longevity of mRNA molecules.

“It is a critical factor in determining the longevity of mRNA molecules (Park et al. 2016; Jalkanen et al. 2014).”

• cites Park et al. 2016 sourceSupported
• cites Jalkanen et al. 2014 sourceSupported

Increasing the G-C fraction of mRNA improves mRNA stability.

“Furthermore, increasing the G-C fraction of mRNA improves mRNA stability (Homma et al. 2016).”

• cites Homma et al. 2016 sourceCould not verify

  The Introduction and Discussion address codon usage, translation speed, and translational accuracy, not mRNA stability. Searches for increased G-C fraction, GC content, and mRNA stability found no passage in our copy supporting the claimed effect. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

The immunogenicity of mRNA is the most challenging obstacle it faces.

“IVT is a low-cost method of mRNA synthesis; however the immunogenicity of mRNA is the most challenging obstacle it faces (Sahin et al. 2014).”

• cites Sahin et al. 2014 sourcePartly supported

  The “Immune-stimulatory activity of IVT mRNA” section calls innate immune activation a major disadvantage for protein-replacement therapies, but an added benefit for vaccination. It does not identify immunogenicity as the most challenging obstacle for mRNA generally; the “Progress in improving mRNA delivery” section also describes substantial delivery challenges. Page numbers are unavailable in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

mRNA vaccines must be stored and carried at extremely low temperatures compared to protein-based vaccinations that can be stored and transported at lower temperatures.

“mRNA vaccines must be stored and carried at extremely low temperatures as compared to protein-based vaccinations that can be stored and transported at lower temperatures (Crommelin et al. 2020).”

• cites Crommelin et al. 2020 sourcePartly supported

  The Introduction describes mRNA vaccine candidates kept at −80 °C and the difficulty of transporting them under those conditions. The storage-conditions table lists frozen storage for three COVID-19 mRNA candidates, but also limited periods at 2–8 °C; the claim's unqualified “must” is too broad. Our copy does not provide the asserted comparison with protein-based vaccines. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Substituting rare codons with identical frequent codons boosts yield.

“Substituting rare codons with identical frequent codons boosts yield as reusing same tRNA speeds up translation due to amino-acylation of tRNAs near ribosomes (Cannarozzi et al. 2010).”

• cites Cannarozzi et al. 2010 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

The 5′ cap shields RNA from exonuclease cleavage.

“The 5′ cap regulates pre-mRNA splicing and nuclear export, works as a protective structure that shields RNA from exonuclease cleavage, and initiates mRNA translation.”

Modifying the mRNA structural elements, particularly the 5′ cap, 5′ and 3′ UTRs, the coding region, and polyadenylation tail, reduces excessive mRNA immunogenicity.

“Modifying the mRNA structural elements particularly the 5′ cap, 5′-and 3′-untranslated regions (UTRs), the coding region, and polyadenylation tail help reduce the excessive mRNA immunogenicity and consistently improve its intracellular stability and translational efficiency.”

ARCA-capped mRNA has twice the translation efficiency of mRNA capped by a conventional cap analog in rabbit reticulocyte lysate.

“When ARCA-capped mRNA was compared to mRNA capped by a conventional cap analog, translation efficiency was twice more than in rabbit reticulocyte lysate (Stepinski et al. 2001).”

• cites Stepinski et al. 2001Not checked — no full text of the source

Deleting the poly(A) region from mRNA results in mRNA being unstable compared to the intact gene.

“Deleting the poly(A) region from mRNA results in mRNA being unstable compared to the intact gene (Whitelaw et al. 1986).”

• cites Whitelaw et al. 1986 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Inserting AU-rich regions into 3′-UTRs ensures fast mRNA breakdown and a short duration of protein expression.

“This effect can be achieved by inserting AU-rich regions into 3′-UTRs ensuring fast mRNA breakdown and a short duration of protein expression (Chen and Shyu 1995).”

• cites Chen and Shyu 1995 sourceNot checked — no full text of the source

The 5′ cap initiates mRNA translation.

“The 5′ cap regulates pre-mRNA splicing and nuclear export, works as a protective structure that shields RNA from exonuclease cleavage, and initiates mRNA translation.”

The length of the poly(A) tail is linked to translation efficiency.

“Several studies have linked the length of the poly(A) tail to translation efficiency (Conry et al. 1995).”

• cites Conry et al. 1995Not matched

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

Avoiding highly stable secondary structures in the 5′ UTR prevents ribosome recruitment and codon recognition.

“(ii) Ribosome recruitment and codon recognition can be prevented by avoiding the existence of highly stable secondary structures”

Modifying the mRNA structural elements, particularly the 5′ cap, 5′ and 3′ UTRs, the coding region, and polyadenylation tail, improves intracellular stability and translational efficiency.

“Modifying the mRNA structural elements particularly the 5′ cap, 5′-and 3′-untranslated regions (UTRs), the coding region, and polyadenylation tail help reduce the excessive mRNA immunogenicity and consistently improve its intracellular stability and translational efficiency.”

• Also stated in: § Introduction · plain

  “This review discussed the iterative optimization and significant modifications of the mRNA structure particularly the 5′ cap, 5′ and 3′UTRs, the coding region, and the poly(A) tail which improve the stability, translation capacity and increase the efficacy of mRNA vaccines to trigger the immune response.”

For mRNA stability and efficient translation, mRNA undergo post-transcriptional changes such as 5′-capping and poly(A).

“For mRNA stability and efficient translation, mRNA undergo post-transcriptional changes such as 5′-capping and poly(A).”

By interacting with RNA-binding proteins, UTRs can influence the rate of mRNA degradation and translation efficiency.

“By interacting with RNA-binding proteins, UTRs can also influence the rate of mRNA degradation and translation efficiency.”

Using enzymes to cap the 5′ terminus of mRNA is more efficient than using cap analogs such as 7-methylguanosine.

“Using enzymes to cap the 5′ terminus of mRNA is more efficient than using cap analogs such as 7-methylguanosine (Jani and Fuchs 2012; Urbina and Morales-Pison 1633).”

• cites Jani and Fuchs 2012Not matched

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

• cites Urbina and Morales-Pison 1633Not matched

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

The most prevalent ARCA enables only the insertion of a nucleotide at the non-methylated guanosine after 3′-O-methylation of base-methylated guanosine.

“The most prevalent ARCA enables only the insertion of a nucleotide at the non-methylated guanosine after 3′-O-methylation of base-methylated guanosine.”

The translational elongation rate and efficiency are affected by neighboring nucleotides and codons.

“The translational elongation rate and efficiency are also affected by the neighboring nucleotides and codons (Bossi and Ruth 1980).”

• cites Bossi and Ruth 1980 sourceNot checked — no full text of the source

5′-capping screens mRNA against exonuclease activity.

“Screening mRNA against exonuclease activity, promoting pre-mRNA splicing, and functioning as binding site of eIF4F heterodimeric translation initiation complex are all important functions of 5′-capping (Izaurralde et al. 1994; Gallie 1991).”

• cites Izaurralde et al. 1994 sourceNot checked — no full text of the source
• cites Gallie 1991 sourceNot checked yet

  Not checked in the free run.

mRNA sequence codon optimization can significantly impact the protein production rate and ribosome bide time.

“mRNA sequence codon optimization can significantly impact the protein production rate and ribosome bide time.”

A functional 5′ cap structure is required for robust mRNA translation.

“A functional 5′ cap structure is required for robust mRNA translation.”

The cap 0 structure of m7GpppN is necessary for effective mRNA translation.

“The cap 0 structure of m7GpppN is necessary for effective mRNA translation.”

Injection of nucleoside-modified mRNA containing 25% thio-uridine and 25% 5-methylcytidine resulted in Epo levels five times greater than untreated animals 2 weeks after injection.

“Kormann et al., demonstrated that 2 weeks following injection of nucleoside-modified mRNA containing 25% thio-uridine and 25% 5-methylcytidine resulted in Epo levels five times greater than that in untreated animals.”

• cites Kormann et al. 2011 sourceNot checked — no full text of the source

The Cap and poly(A) tail at the 5′ and 3′ ends of mRNA are critical for stability of mRNA in the cytosol.

“The Cap and poly(A) tail located at the 5′- and 3′-ends of mRNA are critical for stability of mRNA in the cytosol.”

Substituting the mRNA sequence with rare codons and inserting modified nucleotides most likely alters mRNA structure, translational efficiency, and protein-folding machinery.

“The mRNA structure, translational efficiency, and protein-folding machinery are most likely to be altered by substituting the mRNA sequence with rare codons and inserting modified nucleotides (Zhong et al. 2017).”

• cites Zhong et al. 2017 sourceNot checked — no full text of the source

The rate of codon-dependent translation elongation is a primary factor of mRNA stability.

“Furthermore, the rate of codon-dependent translation elongation has been a primary factor of mRNA stability, codon modification is critical for mRNA stability (Presnyak et al. 2015).”

• cites Presnyak et al. 2015 sourceNot checked yet

  Not checked in the free run.

The employment of anti-reverse cap analogs results in a cap that is in the correct orientation (ARCAs).

“The employment of “anti-reverse” cap analogs results in a cap that is in the correct orientation Anti-Reverse Cap Analog (ARCAs).”

mRNA transcribed in vitro using ARCA has a longer half-life in cultivated cells.

“Furthermore, mRNA transcribed in vitro using ARCA has been found to have a longer half-life in cultivated cells (Grudzien et al. 2006).”

• cites Grudzien et al. 2006 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

The 5′ and 3′ UTRs regulate mRNA stability.

“The 5′- and 3′- UTRs comprising of regulatory sequence elements are known to regulate the mRNA stability.”

• cites Tanguay and Gallie 1996 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Avoiding start codons and non-canonical start codons in the 5′ UTR improves mRNA stability and translation accuracy by preventing disruption of ORF translation.

“The 5′ UTR sequence can be adjusted to improve mRNA stability and translation accuracy by (i) avoiding the presence of start codons and non-canonical start codons in the 5′ UTR which disrupts ORF translation”

Distinct areas of cellular and viral 5′ and 3′ UTRs improve mRNA stability and efficiency of translation.

“Furthermore, distinct areas of cellular and viral 5ʹ- and 3ʹ-UTRs improve mRNA stability and efficiency of translation.”

5′-capping functions as binding site of eIF4F heterodimeric translation initiation complex.

“Screening mRNA against exonuclease activity, promoting pre-mRNA splicing, and functioning as binding site of eIF4F heterodimeric translation initiation complex are all important functions of 5′-capping (Izaurralde et al. 1994; Gallie 1991).”

• cites Izaurralde et al. 1994 sourceNot checked — no full text of the source
• cites Gallie 1991 sourceNot checked yet

  Not checked in the free run.

mRNA needs 5′ and 3′ UTRs around the ORF to increase half-life, vaccine mRNA expression levels, and translation.

“mRNA also need 5′ and 3′ UTRs around the open reading frame (ORF) to increase half-life, vaccine mRNA expression levels, and translation (Conry et al. 1995).”

• cites Conry et al. 1995Not matched

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

Codon composition influences translation efficiency.

“Codon composition is known to influence translation efficiency.”

• cites Kudla et al. 2006 sourceNot checked yet

  Not checked in the free run.

The 5′-UTR/3′-UTR, 5′ cap, poly(A) tail, rare codon and G:C content can be optimized to improve mRNA stability.

“The 5′-UTR/3′-UTR, 5′ cap, the poly(A) tail, a rare codon and G:C content can be optimized to improve mRNA stability.”

The poly(A) tail is important for mRNA stability and translation success.

“The poly(A) tail is important for mRNA stability and translation success (Kudla et al. 2006).”

• cites Kudla et al. 2006 sourceNot checked yet

  Not checked in the free run.

The intensity and specificity of immune responses are expected to be influenced by changes in mRNA structure, translational efficiency, and protein-folding machinery.

“The intensity and specificity of the immune responses are expected to be influenced by these changes.”

• AssumptionRests on an assumption: The intensity and specificity of the immune responses are influenced by changes in mRNA structure, translational efficiency, and protein-folding machinery.

Optimizing mRNA with a GC-rich sequence minimizes mRNA immunogenicity.

“Chemically altering nucleotides, adding poly(A) tails, and optimizing mRNA with a GC-rich sequence are efficient ways to minimize mRNA immunogenicity.”

Adding poly(A) tails minimizes mRNA immunogenicity.

“Chemically altering nucleotides, adding poly(A) tails, and optimizing mRNA with a GC-rich sequence are efficient ways to minimize mRNA immunogenicity.”

With the advancement of lipid nanoparticle techniques, mRNA vaccine stability can be maintained under less rigorous settings.

“However, with the advancement of lipid nanoparticle techniques, mRNA vaccine stability can be maintained under less rigorous settings (Reichmuth et al. 2016).”

• cites Reichmuth et al. 2016 sourceNot checked yet

  Not checked in the free run.

mRNA purification is required after IVT to eliminate immunogenicity.

“mRNA purification is also required after IVT to eliminate immunogenicity.”

High GC concentration reduces the efficacy of protein expression.

“The reduced efficacy of protein expression produced by high GC concentration needs to be addressed (Wang et al. 2021).”

• cites Wang et al. 2021 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Storing and transferring mRNA vaccines to and from warm countries is a major technical and financial challenge.

“Storing and transferring mRNA vaccines to and from warm countries is thus a major technical and financial challenge.”

Chemically altering nucleotides minimizes mRNA immunogenicity.

“Chemically altering nucleotides, adding poly(A) tails, and optimizing mRNA with a GC-rich sequence are efficient ways to minimize mRNA immunogenicity.”

Using structurally modified mRNA may be better for eliciting a rapid antibody response, as highlighted by the COVID-19 mRNA vaccine.

“The COVID-19 mRNA vaccine highlight that using structurally modified mRNA allows for increased dose tolerance and may be better for eliciting a rapid antibody response.”

Using structurally modified mRNA allows for increased dose tolerance, as highlighted by the COVID-19 mRNA vaccine.

“The COVID-19 mRNA vaccine highlight that using structurally modified mRNA allows for increased dose tolerance and may be better for eliciting a rapid antibody response.”

5-methylcytidine (m5C) and pseudouridine (ψ) are the most preferred base-pair alterations because they lower immunogenicity while increasing translation efficiency.

“5-methylcytidine (m5C), and pseudouridine (ψ) are the most preferred base-pair alterations because they lower immunogenicity while increasing translation efficiency (Weng 2020).”

• cites Weng 2020 sourceNot checked — no full text of the source

BNT162b2 and mRNA-1273 used a mixture of altered nucleotide 1mΨ modifications and dsRNA fragment eviction in the mRNA manufacturing procedure to significantly lower innate immune signaling in response to mRNA.

“BNT162b2 (BioNTech/Pfizer) and mRNA-1273 (Moderna) used a mixture of altered nucleotide 1mΨ modifications and dsRNA fragment eviction in the mRNA manufacturing procedure to significantly lower innate immune signaling in response to mRNA (Walsh et al. 2020; Jackson et al. 2020b).”

• cites Walsh et al. 2020 sourceNot checked yet

  Not checked in the free run.

• cites Jackson et al. 2020b sourceNot checked yet

  Not checked in the free run.

Antigen translation occurs in the cytoplasm rather than the nucleus.

“Because antigen translation occurs in the cytoplasm rather than the nucleus; therefore mRNA vaccines have a greater biosafety than DNA vaccines as their antigens are translated into the cytoplasm rather than the nucleus, making it more difficult for the mRNA to integrate into the genome.”

As soon as the sequence encoding the immunogen is available, RNA synthesis may begin on the same platform.

“As soon as the sequence encoding the immunogen is available, RNA synthesis may begin on the same platform.”

The insertion of modified nucleotides into the poly(A) tail by recombinant poly(A) polymerase inhibits deadenylation by poly(A)-specific nucleases.

“The insertion of modified nucleotides into the poly(A) tail by recombinant poly(A) polymerase inhibits deadenylation by poly(A)-specific nucleases (Korner and Wahle 1997).”

• cites Korner and Wahle 1997 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Immunizing against the full S protein rather than just one of its structural units may lead to a better response that is not impacted by genetic drift.

“Therefore, immunizing against the full S protein rather than just one of its structural units may lead to a better response that is not impacted by genetic drift (Liu et al. 2020).”

• cites Liu et al. 2020 sourceNot checked yet

  Not checked in the free run.