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Cited by 0RNA and protein synthesis mechanismsRNA Interference and Gene DeliveryRNA Research and Splicing

Identification of novel 5’ UTR variants for enhanced mRNA translation and vaccine immunogenicity

Open access
Mazunina, Elena P., Dzharullaeva, Alina S., Kleymenov, Denis A., Bykonia, Evgeniia N., Kozlova, Sofia R., Usachev, Evgeny V., Panova, Eugenia A., Konopleva, Maria V. +9 more·2026·Scientific Reports
DOI 10.1038/s41598-026-59586-5
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Abstract

mRNA-based therapeutics represent a highly promising platform for the prevention and treatment of diseases. However, further optimization is required to improve mRNA stability and translational efficiency. The 5' untranslated region (5' UTR) is a critical regulatory element that governs pre-initiation complex assembly and directly influences protein expression levels, making it an important target for rational design. The diverse challenges facing mRNA therapeutics necessitate the development of improved 5' UTR elements adaptable to specific applications. In this study, we evaluated the effects of five different 5' UTR variants, selected based on published data, on firefly luciferase (FLuc) production in cell culture and in an in vivo bioluminescence imaging model in animals. Subsequently, all variants were tested as components of mRNA vaccines against SARS-CoV-2. Our findings demonstrate that the H1.2-TISU and Synth 5' UTRs with mRNA constructs produce increased levels of reporter protein expression in mice, whereas mRNA constructs incorporating the H1.2-TISU 5' UTR demonstrated enhanced humoral immunogenicity compared to the 5' UTR derived from human alpha-globin (HBA) mRNA. Thus, the identified 5' UTRs (H1.2-TISU and Synth) represent promising, in vivo-validated candidates for incorporation into mRNA-based therapeutics and may substantially enhance their efficacy.

AI snapshot

Written from the paper’s full text.

TL;DR

This study compared five 5′ UTRs (HBA reference, H1.2-TISU, Synth, CASP8, Rabb) in mRNA constructs for translation efficiency and SARS-CoV-2 vaccine immunogenicity. H1.2-TISU and Synth supported efficient, prolonged reporter expression in mice, and H1.2-TISU enhanced humoral responses over the HBA 5′ UTR.

Key findings

  • In vitro, DC2.4 cells showed no statistically significant differences among 5′ UTRs; in HEK293T cells at 72 h, CASP8 and Rabb had significantly lower bioluminescence than HBA; in mBMDCs, CASP8 was lower at 3 h and Rabb lower at 3 and 8 h versus HBA (p < 0.05), while Synth was best among experimental constructs versus Rabb and CASP8 at 3, 8, and 24 h.
  • In vivo BLI in mice, all novel 5′ UTRs gave somewhat higher longer-term reporter expression than HBA; at the injection site, CASP8 was higher on day 2 and Rabb higher on day 4 versus HBA (p < 0.05), and total bioluminescence was higher on day 2 for CASP8 and H1.2-TISU and on day 4 for CASP8 and Rabb.
  • In the SARS-CoV-2 mRNA vaccine, antibody titers increased for all constructs after the second immunization; H1.2-TISU was higher than CASP8 at dilutions 9.85 × 10⁻⁵ to 0.025 after V1 and higher than HBA at 4 × 10⁻⁴, and after V2 at 10⁻⁶ H1.2-TISU and Synth were higher than HBA (p < 0.05). AUC comparison showed a significant increase only for H1.2-TISU versus HBA.
  • Structurally, all studied UTRs had similar GC content and MFEI but differed in length and MRL; Synth had the highest MRL value of 7.8.

Methods

  • Five 5′ UTRs were cloned into mRNA constructs with m1Ψ, cap 1 analog, HBA 3′ UTR, and poly(A); mRNAs were either transfected for reporter assays or formulated in LNPs (ALC-0315:DSPC:cholesterol:ALC-0159 at 46.3:9:42.7:1.6; 70–80 nm diameter; PDI 0.08–0.13; encapsulation efficiency 82–92%).
  • In vitro: HEK293T, DC2.4, and murine BMDCs were transfected with Fluc mRNA; luciferase was measured at 3, 8, 24, and 72 h (BMDCs at 3, 8, 24, and 48 h, plus real-time monitoring over 72 h), with BMDCs co-transfected with Actin-Rluc for normalization.
  • In vivo: female BALB/c mice (7–8 weeks, 18 ± 2 g) received single i.m. 5 µg mRNA-LNP doses for BLI (n = 6 per group; intact control n = 3) imaged at 3, 8, 24, 48, 72, 96, and 168 h, or two i.m. 5 µg doses 3 weeks apart for ELISA of RBD-specific IgG on day 14 after each immunization; statistics used Kruskal-Wallis with Dunn’s post hoc test, p < 0.05.

Limitations

  • The paper states that in vivo findings only partially correlate with in vitro data, revealing a pronounced cell-type-specific effect; it also notes higher variability in primary cell cultures due to transfection heterogeneity and potential mRNA quality fluctuations, and that regulatory potential cannot be reliably predicted from nucleotide sequence alone.
  • Evident from the text: no human data are reported; experiments use only female BALB/c mice, small group sizes (n = 6 per group, n = 3 for intact BLI control, n = 4 for PBS ELISA group), a single SARS-CoV-2 S antigen, and one LNP formulation; in vitro assays use transfection rather than LNP delivery.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41598-026-59586-5

Cites

49 of 49 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

14 of 30 cited sources checked · 6 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    In mRNA vaccines, use of different 5′ UTR sequences can substantially enhance immunological properties, including increased antibody titers and improved T-cell responses.

    “In the setting of mRNA vaccines, the use of different 5′ UTR sequences can substantially enhance immunological properties, including increased antibody titers and improved T-cell responses [18,19].”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [18] Reshetnikov, V. et al. Untranslated Region Sequences and the Efficacy of mRNA Vaccines against Tuberculosis. IJMS 25, 888 (2024). source

      Results §2.5 reports that a tuberculosis mRNA vaccine with the TPL 5′ UTR produced more than twice as many IFNγ-secreting cells in immunized mice as one with the mRNA-1273 5′ UTR, supporting the T-cell-response portion. The paper does not report increased antibody titers from changing 5′ UTRs; the antibody finding mentioned in Discussion §3 concerns a different study. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [19] Tang, X. et al. A novel deep generative model for mRNA vaccine development: Designing 5′ UTRs with N1-methyl-pseudouridine modification. Acta Pharmaceutica Sinica B 14, 1814–1826 (2024). source

      The Results section reports substantially higher antibody titers in mice given COVID-19 mRNA vaccines with Smart5UTR-designed 5′ UTRs than with the comparator 5′ UTR. It reports antibody responses, but does not establish the claimed improvement in T-cell responses; the demonstrated finding is also specific to these vaccines in mice. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Premisecitation

    Inclusion of specific untranslated regions is a promising strategy to enhance stability and efficacy of mRNA therapeutics.

    “Recent studies demonstrate that, in addition to the incorporation of modified nucleosides, the inclusion of specific untranslated regions represents a highly promising strategy to enhance the stability and efficacy of mRNA therapeutics [12][13][14][15][16].”
    Quote in the paperSubstantial claim4 of 5 sources checked
    • cites [12] Kirshina, A. et al. Effects of Combinations of Untranslated-Region Sequences on Translation of mRNA. Biomolecules 13, 1677 (2023). source

      The Introduction says UTR sequences play a key role in mRNA stability, and the Conclusions propose selected UTR combinations for therapeutic use because they increase protein expression. But the Results report no significant difference in mRNA copy numbers between groups; improved stability was only hypothesized, not demonstrated for those combinations. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [13] Medjmedj, A. et al. Evaluation of synthetic mRNA with selected UTR sequences and alternative poly(A) tail, in vitro and in vivo. Mol. Therapy Nucleic Acids. 36, 102648 (2025). source
    • cites [14] Volkhin, I. A. et al. Selection of UTRs in mRNA-Based Gene Therapy and Vaccines. Biochem. Mosc. 90, 725–753 (2025). source
    • cites [15] Rubtsova, M. et al. A Luciferase-Based Approach for Functional Screening of 5′ and 3′ Untranslated Regions of the mRNA Component for mRNA Vaccines. Vaccines 13, 530 (2025). source
    • cites [16] Kukushkina-Zvezdova, A. A. et al. A comprehensive analysis of the use of nucleoside analogues in RNA therapeutics. J. Transl Med. https://doi.org/10.1186/s12967-026-08083-4 (2026). source

      In “Synthesis of mRNA with region-specific modifications” (page unavailable), the review says that optimizing mRNA secondary structure for stability and translatability takes UTR structures into account. It does not specifically say that including particular untranslated regions enhances therapeutic stability and efficacy; its proposed strategy concerns sequence or structural optimization and region-specific nucleoside modifications. (LLM, conf=0.81)

      Experimental — accuracy not yet measured
  • Premisecitation

    Untranslated regions harbor regulatory elements that influence translational efficiency and mRNA stability within cells.

    “The untranslated regions harbor regulatory elements that influence translational efficiency and mRNA stability within cells [10,11].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [10] Leppek, K., Das, R. & Barna, M. Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them. Nat. Rev. Mol. Cell. Biol. 19, 158–174 (2018). source
    • cites [11] Jia, J., Yao, P., Arif, A. & Fox, P. L. Regulation and dysregulation of 3′UTR-mediated translational control. Curr. Opin. Genet. Dev. 23, 29–34 (2013). source

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

  • Premisecitation

    The rate of mRNA degradation also plays a pivotal role in reporter protein accumulation.

    “The rate of mRNA degradation also plays a pivotal role in reporter protein accumulation.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [36] Jia, L. et al. Decoding mRNA translatability and stability from the 5′ UTR. Nat Struct Mol Biol 27, 814–821 (2020). source

      The Results report a positive correlation between reporter mRNA half-life and GFP intensity (Fig. 2c), linking mRNA stability with reporter protein accumulation. But the paper chiefly demonstrates that translation affects mRNA stability; it does not establish that the degradation rate itself plays a pivotal causal role in protein accumulation. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [37] Bicknell, A. A. et al. Attenuating ribosome load improves protein output from mRNA by limiting translation-dependent mRNA decay. Cell Reports 43, 114098 (2024). source
  • Findingown data

    In mBMDCs, experimental 5′ UTRs provided high luciferase activity comparable to control HBA 5′ UTR.

    “The experimental 5′ UTRs provided high luciferase activity comparable to that of the control HBA 5′ UTR.”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 18
  • May combine two claims. 20

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Variations in reporter protein accumulation within tissues may also be influenced by other factors, including differences in elongation rates, physicochemical properties of LNPs, and kinetics of endosomal escape into cytosol.

    “Variations in reporter protein accumulation within tissues may also be influenced by other factors, including differences in elongation rates, the physicochemical properties of LNPs, and the kinetics of endosomal escape into the cytosol [33].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [33] Xu, X., Cui, L., Zhang, Y. & Gu, J. Deciphering the biological fate of mRNA-LNP-based biologics: A perspective from tissue to intracellular distribution. Acta Pharm. Sinica B. S2211383525007737 https://doi.org/10.1016/j.apsb.2025.11.023 (2025). source

      The section on factors influencing expressed-protein distribution links tissue-level differences to LNP physicochemical properties and tissue-specific translational efficiency. The endosomal-escape section links escape efficiency to protein expression, but our copy does not identify differences in elongation rates as a cause of reporter-protein accumulation or establish that endosomal-escape kinetics explain variation between tissues. No page numbers are available in the indexed passages. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Expression profiles of these factors vary across different cells and tissues, and their interactions with 5′ UTR constitute a vital endogenous mechanism for precise cell- or tissue-specific mRNA regulation.

    “The expression profiles of these factors vary across different cells and tissues, and their interactions with the 5′ UTR constitute a vital endogenous mechanism for achieving precise cell-or tissue-specific mRNA regulation [32].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [32] Mao, L., Tian, Y., Qian, K., Song, Y. & RNALens Study on 5’ UTR Modeling and Cell-Specificity. Preprint at. https://doi.org/10.1101/2025.07.20.665722 (2025). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Several synthetic 5′ UTR sequences have demonstrated strong performance.

    “However, several synthetic sequences have also demonstrated strong performance [14,19].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [14] Volkhin, I. A. et al. Selection of UTRs in mRNA-Based Gene Therapy and Vaccines. Biochem. Mosc. 90, 725–753 (2025). source
    • cites [19] Tang, X. et al. A novel deep generative model for mRNA vaccine development: Designing 5′ UTRs with N1-methyl-pseudouridine modification. Acta Pharmaceutica Sinica B 14, 1814–1826 (2024). source

  • May combine two claims.

    Premisecitation

    The 5′ UTRs of highly expressed natural genes such as HBA1 and HBB provide high translational activity and mRNA stability.

    “These criteria are met by the 5′ UTRs of highly expressed natural genes such as HBA1 and HBB, encoding α-and β-globin, respectively, which provide high translational activity and mRNA stability [21,22].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [21] Xia, X. Detailed Dissection and Critical Evaluation of the Pfizer/BioNTech and Moderna mRNA Vaccines. Vaccines 9, 734 (2021). source

      Section 3.3 identifies the 5′ UTR of the highly expressed α-globin gene as a strategy for facilitating translation. Section 3.5 says α-globin mRNA is translated efficiently and that globin 5′ and 3′ UTRs can confer mRNA stability. It does not specifically establish high translational activity for the HBB 5′ UTR; its discussion of β-globin translation-related design concerns the 3′ UTR. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [22] Jung, S.-J. et al. RNA stability enhancers for durable base-modified mRNA therapeutics. Nat Biotechnol https://doi.org/10.1038/s41587-025-02891-7 (2025). source

  • May combine two claims.

    Premisecitation

    mRNAs characterized by high translation initiation rates exhibit high ribosomal loading, which increases frequency of ribosome collisions, leading to accelerated mRNA decay and reduced product yield.

    “On the other hand, mRNAs characterized by high translation initiation rates exhibit high ribosomal loading, which increases the frequency of ribosome collisions, leading to accelerated mRNA decay and, consequently, reduced product yield [37].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [37] Bicknell, A. A. et al. Attenuating ribosome load improves protein output from mRNA by limiting translation-dependent mRNA decay. Cell Reports 43, 114098 (2024). source

      The Results link faster initiation to higher ribosome load and faster mRNA decay in one tested mRNA subset; the Discussion links high load to lower protein output. The Discussion also says denser ribosome loading is associated with more collisions, but presents collisions as a possible mechanism for the faster decay, not an established cause in these mRNAs. The citing sentence states that causal link without the paper’s qualification. Page numbers are unavailable in our copy. (LLM, conf=0.90)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    mRNAs harboring H1.2-TISU and Synth 5′ UTRs demonstrated translational efficiency comparable to HBA control, whereas CASP8 and Rabb 5′ UTR-containing mRNAs exhibited lower reporter expression.

    “Notably, mRNAs harboring the H1.2-TISU and Synth 5′ UTRs demonstrated translational efficiency comparable to that of the HBA control, whereas CASP8 and Rabb 5′ UTRcontaining mRNAs exhibited lower reporter expression.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At injection site, bioluminescence intensity was significantly higher on day 2 for mRNA-LNP with CASP8 5′ UTR and on day 4 for constructs containing Rabb compared to HBA control (p < 0.05).

    “At the injection site, bioluminescence intensity was significantly higher on day 2 for mRNA-LNP with the CASP8 5′ UTR and on day 4 for constructs containing Rabb (p < 0.05) compared to the HBA control (Fig. 3b).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    High levels of bioluminescence were detected in liver during first 24 h after injection in all groups.

    “It is noteworthy that high levels of bioluminescence were detected in the liver during the first 24 h after injection in all groups (Fig. 3d).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Continuous luminescence measurements revealed similar expression kinetics for all five mRNA constructs over 72 h, with peak luciferase activity at 13-16 h post-transfection followed by gradual decline.

    “Continuous luminescence measurements revealed similar expression kinetics for all five mRNA constructs over the extended observation period, with peak luciferase activity detected at 13-16 h post-transfection, followed by a gradual decline (Fig. 2d).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    H1.2-TISU and Synth, despite similar efficacy, differ in their length, MFEI, and MRL values.

    “A detailed examination of the most potent 5′ UTRs tested here reveals that H1.2-TISU and Synth, despite showing similar efficacy, differ in their length, MFEI, and MRL values.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    mRNAs featuring Rabb and CASP8 5′ UTRs enhanced luciferase biosynthesis in murine model, but resulted in lower reporter protein levels in HEK293T and mBMDC cell lines compared to HBA 5′ UTR.

    “While mRNAs featuring the Rabb and CASP8 5′ UTRs also enhanced luciferase biosynthesis in the murine model, they resulted in lower reporter protein levels in HEK293T and mBMDC cell lines compared to the HBA 5′ UTR (Table 2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After first immunization, antibody titers in H1.2-TISU group were significantly higher compared to CASP8 group at serum dilutions ranging from 9.85 × 10−5 to 0.025, and higher than HBA group at dilution of 4 × 10−4 (p < 0.05).

    “Analysis at individual serum dilutions showed that, after the first immunization, antibody titers in the H1.2-TISU group were significantly higher compared to the CASP8 group at serum dilutions ranging from 9.85 × 10⁻⁵ to 0.025, and higher than the HBA group at a dilution of 4 × 10⁻⁴ (p < 0.05, Fig. 4a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    All studied UTRs possess similar GC content and MFEI parameters, differing primarily in length and MRL values.

    “Our results indicate that all studied UTRs possess similar GC content and MFEI parameters, differing primarily in length and MRL values.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Increased total bioluminescence was detected on day 2 for mRNAs containing CASP8 and H1.2-TISU 5′ UTRs and on day 4 for CASP8 and Rabb constructs relative to HBA (p < 0.05).

    “Similarly, increased total bioluminescence was detected on day 2 for mRNAs containing CASP8 and H1.2-TISU 5′ UTRs and on day 4 for CASP8 and Rabb constructs relative to HBA (p < 0.05).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Changes in total bioluminescence and signal intensity at injection site followed similar trends.

    “Changes in total bioluminescence and signal intensity at the injection site followed similar trends (Fig. 3a,b).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The average particle diameter of the mRNA-LNPs was 70-80 nm, with a polydispersity index of 0.08-0.13.

    “The average particle diameter was 70-80 nm, with a polydispersity index of 0.08-0.13.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    The 5′ UTR may contain additional regulatory elements such as uAUGs, uORFs, G-quadruplexes, 5′ TOP, and recognition sites for RNA-binding proteins and small RNAs that modulate translation rates.

    “The 5′ UTR may also contain additional regulatory elements, such as upstream AUG codons (uAUGs) and upstream open reading frames (uORFs), G-quadruplexes, a terminal oligopyrimidine tract (5′ TOP), and recognition sites for RNA-binding proteins and small RNAs that modulate translation rates [14].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [14] Volkhin, I. A. et al. Selection of UTRs in mRNA-Based Gene Therapy and Vaccines. Biochem. Mosc. 90, 725–753 (2025). source

  • Classified as probably not a substantial claim.

    Premisecitation

    Efficiency of eukaryotic 5′ UTRs depends on length, stability, secondary structure features and positioning, nucleotide composition, and other factors.

    “The efficiency of eukaryotic 5′ UTRs depends on their length, stability, secondary structure features and positioning, nucleotide composition, and other factors [20].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [20] Babendure, J. R., Babendure, J. L., Ding, J. H. & Tsien, R. Y. Control of mammalian translation by mRNA structure near caps. RNA. 12, 851–861 (2006). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Energy corrections for incorporation of N1-methylpseudouridine (m1Ψ) were applied during analysis.

    “Energy corrections for the incorporation of N1methylpseudouridine (m 1 Ψ) were applied during the analysis.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    An extensively stable secondary structure with high GC content in the 5′ UTR, particularly in the 5′-proximal region, tends to decrease translation efficiency.

    “It is well established that an extensively stable secondary structure with high GC content in the 5′ UTR -particularly in the 5′-proximal region -tends to decrease translation efficiency [10,34].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [10] Leppek, K., Das, R. & Barna, M. Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them. Nat. Rev. Mol. Cell. Biol. 19, 158–174 (2018). source

      Not checked in the free run.

    • cites [34] Eisenhut, P. et al. Systematic use of synthetic 5′-UTR RNA structures to tune protein translation improves yield and quality of complex proteins in mammalian cell factories. Nucleic Acids Research 48, e119–e119 (2020). source

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

  • May combine two claims.

    Interpretationown data

    Our findings in vivo only partially correlate with in vitro data, revealing a pronounced cell-type-specific effect.

    “It is crucial to highlight that our findings in vivo only partially correlate with the in vitro data, revealing a pronounced cell-type-specific effect.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    The 5′ UTR plays a key role in translation initiation and pre-initiation complex assembly.

    “In this context, the 5′ UTR is of particular interest, as it plays a key role in translation initiation and pre-initiation complex assembly [17].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [17] Hinnebusch, A. G., Ivanov, I. P. & Sonenberg, N. Translational control by 5′-untranslated regions of eukaryotic mRNAs. Science 352, 1413–1416 (2016). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    RNA secondary structure elements can facilitate higher-order interactions that promote assembly of intermolecular complexes involving RBPs and regulatory RNA molecules, making overall impact of secondary structure multifaceted.

    “However, RNA secondary structure elements can also facilitate higher-order interactions that promote the assembly of intermolecular complexes involving RBPs and regulatory RNA molecules [10], making the overall impact of secondary structure multifaceted.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Leppek, K., Das, R. & Barna, M. Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them. Nat. Rev. Mol. Cell. Biol. 19, 158–174 (2018). source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    It is widely suggested that cell-type specificity of mRNA translation is primarily dictated by 3′ UTR, but emerging evidence points to significant regulatory role of 5′ UTR as well.

    “While it is widely suggested that the cell-type specificity of mRNA translation is primarily dictated by the 3′ UTR [31], emerging evidence points to a significant regulatory role of the 5′ UTR as well.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [31] Castillo-Hair, S. et al. Optimizing 5’UTRs for mRNA-delivered gene editing using deep learning. Nat Commun 15, 5284 (2024). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    RNA integrity was verified by denaturing PAGE.

    “RNA integrity was verified by denaturing PAGE.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All animal procedures were approved by the Local Ethics Committee of the National Research Centre for Epidemiology and Microbiology named after the Honorary Academician N.F.Gamaleya (Protocol No. 94, May 20, 2025) and conducted in accordance with ARRIVE 2.0 guidelines and national regulations on the use of laboratory animals.

    “All procedures were approved by the Local Ethics Committee of the National Research Centre for Epidemiology and Microbiology named after the Honorary Academician N.F.Gamaleya (Protocol No. 94, May 20, 2025) and conducted in accordance with ARRIVE 2.0 guidelines and national regulations on the use of laboratory animals.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Such cross-talk can be realized through formation of long-range RNA-RNA interactions (secondary and tertiary structures) or mediated by components of translational machinery, such as RNA-binding proteins and factors that form mRNA closed-loop structure.

    “Such cross-talk can be realized through the formation of long-range RNA-RNA interactions (secondary and tertiary structures) or mediated by components of the translational machinery, such as RNA-binding proteins and factors that form the mRNA closed-loop structure [35].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [35] Alekhina, O., Terenin, I., Dmitriev, S. & Vassilenko, K. Functional Cyclization of Eukaryotic mRNAs. IJMS 21, 1677 (2020). source

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

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All experiments were repeated at least three times, and mean values ± SD were calculated.

    “All experiments were repeated at least three times (including those with different cell passages), and mean values ± SD were calculated.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    Our results suggest their potential utility in both mRNA therapeutics and mRNA vaccines.

    “Our results suggest their potential utility in both mRNA therapeutics and mRNA vaccines -the two key areas for the application of mRNA-based drugs [38].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [38] Rohner, E., Yang, R., Foo, K. S., Goedel, A. & Chien, K. R. Unlocking the promise of mRNA therapeutics. Nat Biotechnol 40, 1586–1600 (2022). source

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

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Sequence integrity of constructs was confirmed by Sanger sequencing.

    “Sequence integrity was confirmed by Sanger sequencing.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Active translation is thought to protect mRNA from degradation.

    “On one hand, active translation is thought to protect mRNA from degradation [36].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [36] Jia, L. et al. Decoding mRNA translatability and stability from the 5′ UTR. Nat Struct Mol Biol 27, 814–821 (2020). source

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

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    For each construct, three biological and three technical replicates were set up at each time point.

    “For each construct, three biological and three technical replicates were set up at each time point.”
    Quote in the paperNot a substantial claim
32 of 32

  • The paper relies on this without support.

    It remains impossible to reliably predict the regulatory potential of a given 5′ UTR based solely on its nucleotide sequence.

    “Importantly, it remains impossible to reliably predict the regulatory potential of a given 5′ UTR based solely on its nucleotide sequence.”

  • The paper relies on this without support.

    Mouse immunogenicity data are predictive of human immunogenicity.

    “Mice were immunized twice by intramuscular injection into the thigh at a dose of 5 μg per mouse, with a 3week interval between administrations.”

  • The paper relies on this without support.

    The effect of a 5' UTR on luciferase reporter translation is representative of its effect on the SARS-CoV-2 S protein translation.

    “Experimental design for comparison of 5′ UTR activity (Synth, CASP8, H1.2-TISU, Rabb, and HBA) in mRNA formulations.(a) In vitro and in vivo study design using FLuc-encoding mRNA.”
3 of 3

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Post-boost antibody result points to the bioluminescence figure

    Minor

    The post-boost antibody comparison cites Fig. 3b, but the paper identifies Fig. 3b as injection-site bioluminescence and Fig. 4b as the post-boost antibody results.

  • First cell-assay time point is reported as both 3 and 4 hours

    Minor

    The experimental-design figure says luciferase was measured at 4 hours in both cell lines and primary BMDCs, whereas the results and methods identify the first measurement as 3 hours.

  • Animal-study ethics approvals differ

    Minor

    The animal-studies methods give protocol No. 94, approved May 20, 2025, while another passage in the supplied text gives protocol #41 dated 04/06/2023. The text does not reconcile these as separate approvals.

3 of 3

Cites

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Related

Authors
Mazunina, E. P., Dzharullaeva, A. S., Kleymenov, D. A., Bykonia, E. N., Kozlova, S. R., Usachev, E. V., Panova, E. A., Konopleva, M. V., Telegina, D. V., Kukushkina-Zvezdova, A. A., Zherebtsova, A. A., Kukushkin, I. S., Logunov, D. Y., Ivanov, R. A., Reshetnikov, V. V., Dmitriev, S. E., & Gushchin, V. A.
Affiliations
  • Ministry of Health of the Russian Federation (RU)
  • Lomonosov Moscow State University (RU)
  • Sirius University of Science and Technology (RU)
  • Sechenov University (RU)
Corresponding author
  • Reshetnikov, Vasiliy V.
  • Dmitriev, Sergey E.
  • Gushchin, Vladimir A.
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2026-06-29
Volume
16
Issue
1
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-026-59586-5
OpenAlex
W7166510990
Cited by
0
References
4
FWCI
0.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
The study was supported by the grant of the state program of the «Sirius» Federal Territory «Scientific and technological development of the «Sirius» Federal Territory» (Agreement No. 3-03 on 18 February 2025)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
Keywords
5' untranslated region, mRNA vaccines, SARS-CoV-2, mRNA translation efficiency, mRNA stability, firefly luciferase reporter, in vivo bioluminescence imaging, humoral immunogenicity, mRNA therapeutics

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-05.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-05.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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