Journal: Frontiers in immunology

Article Title: Discovery and development of a safe and efficient COVID-19 mRNA vaccine, STP2104, using a novel capping library screening method.

doi: 10.3389/fimmu.2025.1571713

Figure Lengend Snippet: FIGURE 1 Evaluation of mRNA yield, capping efficiency, and potency of reporter genes using capping library screening. (a) SmartCap® (SC) library composition with ribose and base modification. In synthesized eGFP mRNA, (b) IVT products on 1% agarose gel, (c) capping efficiency of several 5′-cap analogues, (d) naked eGFP mRNA transfection-mediated fluorescence in HEK293T and Huh7 were evaluated. (e) In hEPO mRNA synthesis, mRNA yield, and potency were determined via hEPO ELISA assay in HEK293T and RD cell culture media. (f) In fLUC mRNA synthesis, yield, and potency were determined via luciferase assay in HEK293T and C2C12 cell lysate. (h) Female BALB/c mice were intramuscularly injected with PBS or a 5 mg dose of fLUC (Capping with SmartCap® library) mRNA LNPs. Luminescence was determined via IVIS (IVIS spectrum, PerkinElmer) up to 6 days p.i. Luminescence image from the whole body was measured over time for up to 6 days p.i. (h) Total flux of the whole body was plotted over time. Statistical analyses were performed using one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, FF ns: no significant).

Article Snippet: For mRNA transfection, HEK293T cells (1 × 106 cells/well) were plated in 6-well plates and cultured in GibcoTM Opti-MEMTM Reduced Serum Medium (Thermo Fisher Scientific) for 24 h. After 24 h of transfection, the fluorescence expressed by eGFP and hEPO in the cell cultures was analyzed by using an EVOS M5000 Microscope (Thermo Fisher Scientific) with a 10X scope and a Human Erythropoietin/EPO ELISA Kit (BioTechne® R&D System, Quantikine DEPRU0, Minneapolis, MN, USA), respectively.

Techniques: Library Screening, Synthesized, Agarose Gel Electrophoresis, Analogues, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Luciferase, Injection