Journal: bioRxiv

Article Title: 3’ UTR Insertion of a Directed-Evolved RNA Element for Enhanced Translation

doi: 10.64898/2026.05.07.723449

Figure Lengend Snippet: (A), Schematic of P51 elements inserted at five sites in the CMV-EGFP reporter 3’UTR. (B), The results of P51 elements inserted at five sites in the CMV-EGFP reporter 3’UTR (Moderna mRNA-1273 3’ UTR) was evaluated by fluorescence spectroscopy in HEK293T cells transfected with the respective engineered plasmids. Scale bar = 1000 µm. (C), Measurement of EGFP fluorescence enhancement mediated by P51 elements inserted at five sites in the CMV-EGFP reporter 3’UTR (Moderna mRNA-1273 3’UTR). Data are normalized to the CMV-EGFP reporter 3’UTR lacking P51 insertions (Ctrl) and presented as mean ± SEM (n = 3). **P < 0.01, two-tailed Student’s t-test. (D), Structures of the P51 element and its four truncations. RNA secondary structures were predicted using the RNAfold WebServer. (E), Schematic of P51 element and its four truncations inserted in the CMV-EGFP reporter 3’UTR. (F), The results of P51 element and its four truncations inserted in the CMV-EGFP reporter 3’UTR (Moderna mRNA-1273 3’UTR) was evaluated by fluorescence spectroscopy in HEK293T cells transfected with the respective engineered plasmids. Scale bar = 1000 µm. (G), Measurement of EGFP fluorescence enhancement mediated by P51 element and its four truncations inserted in the CMV-EGFP reporter 3’UTR (Moderna mRNA-1273 3’UTR). Data are normalized to the CMV-EGFP reporter 3’UTR lacking P51 or P51-truncation insertions (Ctrl) and presented as mean ± SEM (n = 3). ***P < 0.001, ****P < 0.0001, two-tailed Student’s t-test. (H), Schematic diagram of inserting the P51 element or its truncated forms into the pCAG 3’UTR. There were 121 bases between the insertion site and polyA. After transcription, through the mRNA closed-loop translation model, P51 or its truncated forms was spatially close to the 5’ region of mRNA. Data are normalized to the reporter 3’UTR lacking P51 or its truncated forms insertions (Ctrl). (I), P51t2 and P51t3 further enhanced the translation of EGFP in C2C12. Scale bar = 1000 µm. (J), P51t2 and P51t3 further enhanced the translation of Fluc in C2C12.The results of the dual-luciferase reporter system from the C2C12 cells transfected with the plasmid at 48h post transfection. Data are presented as mean ±SEM n = 4. Two-tailed Student’s t-test. ***P < 0.001, ****P < 0.0001. (K), P51t2 and P51t3 further enhanced the translation of uDys in C2C12. Western blot data from the C2C12 cells transfected with the plasmid at 48h post transfection. (L), Quantitative heat map of the experiment of figure (k). n=5. (M), The RT-qPCR results of uDys mRNA. Data are presented as mean ±SEM n = 4. Two-tailed Student’s t-test. ns, not significant.

Article Snippet: In clinically relevant models, insertion of P51t3 into an mRNA containing Moderna mRNA-1273 UTRs increased spike protein expression 2–3-fold in C2C12 cells ( ).48 Similarly, in constructs mimicking BioNTech BNT162b2 UTRs, P51t3 boosted Fluc translation nearly 2-fold ( ).

Techniques: Fluorescence, Spectroscopy, Transfection, Two Tailed Test, Luciferase, Plasmid Preparation, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: 3’ UTR Insertion of a Directed-Evolved RNA Element for Enhanced Translation

doi: 10.64898/2026.05.07.723449

Figure Lengend Snippet: (A), Schematic of P51 elements inserted at five sites in the CMV-Fluc reporter 3’UTR. (B), The results of P51 elements inserted at five sites in the CMV-Fluc reporter 3’UTR (Moderna mRNA-1273 3’UTR) was evaluated by dual-luciferase assay in HEK293T cells transfected with the respective engineered plasmids. Data are normalized to the CMV-Fluc reporter 3’ UTR lacking P51 insertions (Ctrl) and presented as mean ± SEM (n = 3). ****P < 0.0001, two-tailed Student’s t-test. (C), Schematic of P51 element and its four truncations inserted in the CMV-Fluc reporter 3’UTR. (D), The results of P51 element and its four truncations inserted in the CMV-Fluc reporter 3’UTR (Moderna mRNA-1273 3’UTR) was evaluated by dual-luciferase assay in HEK293T cells transfected with the respective engineered plasmids. Data are normalized to the CMV-Fluc reporter 3’UTR lacking P51 or P51-truncation insertions (Ctrl) and presented as mean ± SEM (n = 3). **P < 0.01,***P < 0.001, two-tailed Student’s t-test. (E), P51t3 is inserted at different sites of B2M 3’UTR. (F), P51t3 enhanced the translation of Fluc (B2M 3’UTR) in HEK293T. The results of the dual-luciferase reporter system from the HEK293T cells transfected with the plasmid at 48h post transfection. Data are normalized to the reporter 3’UTR lacking P51t3 insertions (Ctrl) and presented as mean ±SEM n = 5. Two-tailed Student’s t-test. **P < 0.01, ****P < 0.0001. (G), P51t3 is inserted at different sites of TMSB10 3’UTR. (H), P51t3 enhanced the translation of Fluc (TMSB10 3’UTR) in HEK293T. The results of the dual-luciferase reporter system from the HEK293T cells transfected with the plasmid at 48h post transfection. Data are normalized to the reporter 3’UTR lacking P51t3 insertions (Ctrl) and presented as mean ±SEM n = 5. Two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ****P < 0.0001.

Article Snippet: In clinically relevant models, insertion of P51t3 into an mRNA containing Moderna mRNA-1273 UTRs increased spike protein expression 2–3-fold in C2C12 cells ( ).48 Similarly, in constructs mimicking BioNTech BNT162b2 UTRs, P51t3 boosted Fluc translation nearly 2-fold ( ).

Techniques: Luciferase, Transfection, Two Tailed Test, Plasmid Preparation

Journal: bioRxiv

Article Title: 3’ UTR Insertion of a Directed-Evolved RNA Element for Enhanced Translation

doi: 10.64898/2026.05.07.723449

Figure Lengend Snippet: (A), Structures of the P51 element and its truncations P51t2 and P51t3. Blue circles denote evolutionary sites; gray circles indicate potential MRE sites. RNA secondary structures were predicted using RNAfold WebServer and rendered using RNAcanvas. (B), Predicted MRE landscape of the Moderna mRNA-1273 3’UTR Site2 upon insertion of P51 or its truncations. Target scores are displayed as heat maps. MRE analysis was performed using miRDB. (C), Predicted MRE landscape of the pCAG 3’UTR L1 upon insertion of SINEB2 element or P51 element or P51-truncations. Target scores are displayed as heat maps. MRE analysis was performed using miRDB. (D), Predicted MRE landscape of the Moderna mRNA-1273 3’UTR Site3 upon insertion of P51 or its truncations. Target scores are displayed as heat maps. MRE analysis was performed using miRDB.

Article Snippet: In clinically relevant models, insertion of P51t3 into an mRNA containing Moderna mRNA-1273 UTRs increased spike protein expression 2–3-fold in C2C12 cells ( ).48 Similarly, in constructs mimicking BioNTech BNT162b2 UTRs, P51t3 boosted Fluc translation nearly 2-fold ( ).

Techniques:

Journal: bioRxiv

Article Title: 3’ UTR Insertion of a Directed-Evolved RNA Element for Enhanced Translation

doi: 10.64898/2026.05.07.723449

Figure Lengend Snippet: (A), Schematic of P51 element inserted at three sites within the 3’UTR of IVT mRNA. (B), The results of P51 element inserted at three sites within the 3’UTR of IVT mRNA was evaluated by dual-luciferase assay in HEK293T cells transfected with the respective mRNAs. Data are normalized to the IVT mRNA 3’UTR lacking P51 insertions (Ctrl) and presented as mean ± SEM (n = 3). **P < 0.01, ****P < 0.0001, two-tailed Student’s t-test. (C), Schematic of P51 element or its truncations inserted at Site2 and Site3 within the 3’UTR of IVT mRNA. (D), The results of P51 element or its truncations inserted at Site2 and Site3 within the 3’UTR of IVT mRNA was evaluated by dual-luciferase assay in HEK293T cells transfected with the respective mRNAs. Data are normalized to the IVT mRNA 3’UTR lacking P51 insertions (Ctrl) and presented as mean ± SEM (n = 3). (E), The results of P51t3 element inserted at Site3 within the 3’UTR of IVT mRNA was evaluated by dual-luciferase assay in C2C12 and HEK293T cells transfected with 50 ng (gray) or 100 ng (blue) of the respective mRNAs. Data are normalized to the IVT mRNA 3’UTR lacking P51t3 insertions (Ctrl) and presented as mean ± SEM (n = 3). ***P < 0.001, ****P < 0.0001, two-tailed Student’s t-test. (F), Schematic of P51 elements inserted at three sites within the 3’UTR (Moderna mRNA-1273 3’UTR) of IVT EGFP mRNA. (G), (H), The results of P51 elements inserted at three sites within the 3’UTR (Moderna mRNA-1273 3’UTR) of IVT EGFP mRNA was evaluated by fluorescence spectroscopy in HEK293T cells transfected with the respective mRNAs. Scale bar = 1000 µm. EGFP data are normalized to the IVT mRNA 3’UTR lacking P51t3 insertions (Ctrl) and presented as mean ± SEM (n = 3). ****P < 0.0001, two-tailed Student’s t-test. (I), Schematic of P51t3 element inserted at Site3 within the 3’UTR of IVT Moderna mRNA-1273. (J), The results of P51t3 element inserted at Site3 within the 3’UTR of IVT Moderna mRNA-1273 was evaluated by western blot assay in HEK293T cells transfected with the mRNAs. Data are normalized to the IVT mRNA 3’UTR lacking P51t3 insertions (Ctrl) and presented as mean ± SEM (n = 3). **P < 0.01, two-tailed Student’s t-test. (K), Schematic of P51t3 elements inserted at four sites within the 3’UTR (BioNTech/Pfizer BNT-162b2 3’UTR) of IVT Fluc mRNA. (L), The results of P51t3 elements inserted at four sites within the 3’UTR (BioNTech/Pfizer BNT-162b2 3’UTR) of IVT Fluc mRNA was evaluated by dual-luciferase assay in HEK293T cells transfected with the respective mRNAs. Data are normalized to the IVT mRNA 3’UTR lacking P51t3 insertions (Ctrl) and presented as mean ± SEM (n = 3). ****P < 0.0001, two-tailed Student’s t-test. (M), Schematic of in vivo luminescence quantification. Fluc mRNAs were formulated in LNPs and equal molar quantities of mRNA-LNP complexes were administered through IM injection to male 6-8 weeks C57BL/6J mice. (N), (O), Exemplary in vivo luminescence images of mice treated with Fluc mRNA at 4, 24 and 48 h post mRNA–LNP complex administration. Color scale of the heatmaps, radiance (photons s−1 cm−2 sr−1). Luminescence was measured by integration of total flux for each mouse. Data are normalized to the mice of Ctrl group (IVT mRNA 3’UTR lacking P51t3 insertions-LNP-treated) and presented as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, two-tailed Student’s t-test.

Article Snippet: In clinically relevant models, insertion of P51t3 into an mRNA containing Moderna mRNA-1273 UTRs increased spike protein expression 2–3-fold in C2C12 cells ( ).48 Similarly, in constructs mimicking BioNTech BNT162b2 UTRs, P51t3 boosted Fluc translation nearly 2-fold ( ).

Techniques: Luciferase, Transfection, Two Tailed Test, Fluorescence, Spectroscopy, Western Blot, In Vivo, Injection