mRNA produced by VSW-3 RNAP has high-level translation efficiency with low inflammatory stimulation

Figures

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  Fig. 1. Expression of GFP and Flu mRNA in HEK239T and Hela cells. (A) Green fluorescence signals were detected by fluorescence microscopy at various time points after transfection. (B) Flow cytometry was applied to detect the green fluorescence signals of HEK293T cells at various time points after transfection of mRNA, and median fluorescence intensity was determined. (C) Twenty hours after transfection of GFP mRNA into HEK293T and Hela cells, the ratios of GFP-positive cells were determined by flow cytometry. (D) Twenty hours after transfection of GFP mRNA into HEK293T (n = 14) and Hela (n = 10) cells, the MFI of GFP-positive cells was calculated. (E) The Flu mRNA produced by VSW-3 or T7 RNAP were transfected into HEK293T cells, and relative light unit (RLU) of luciferase was determined at different time points. (F) Flu mRNA transcribed by VSW-3 or T7 RNAP were transfected into HEK239T (n = 8) and Hela (n = 5) cells. The RLU were detected at 20 h after transfection of mRNA. Data are shown as mean ± SD. n referred to the numbers of batches of mRNA transcripts prepared by VSW-3 or T7 RNAP. Paired t-test was used. ∗∗p < 0.01, ∗∗∗p < 0.001.

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  Fig. 2. The expression levels of Flu mRNA produced by VSW-3 or T7 RNAP in mice. (A) Schematic diagram of Flu mRNA-LNP preparation. (B) Size distribution of Flu mRNA-LNP particles in VSW-3 group and T7 group. (C) Flu mRNA-LNP prepared by VSW-3 or T7 were injected into mice via tail vein. Animals in mock group were injected with normal saline. Six hours after injection, the mice were anesthetized for imaging, and regions of interest (ROI) were determined by amiview software. (D) The Luciferase intensity of the VSW-3 and the T7 group were compared. Data are shown as mean ± SD. Unpaired t-test was used. ∗∗p < 0.01, n = 5.

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  Fig. 3. The dsRNA byproducts in mRNA produced by the VSW-3 or T7 RNAP. (A) The IVT buffer of VSW-3 and NEB (#M0251S) were used to produce GFP mRNA which was transfected into HEK293T cells, and the MFI of GFP-positive cells was determined by flow cytometry. (B) Twenty hours after transfection, the expression levels of RIG-I, MDA5, and IFN-β in HEK293T cells were determined by qPCR. The negative control group was transfection agent treated only. (C) Dot blot analysis of dsRNA of GFP, Flu and F9 mRNA produced by VSW-3 or T7 RNAP. (D) Dot blot analysis of dsRNA in GFP mRNA with or without HPLC purification. For the dot blot analysis, 0.1 μg of RNA was loaded for each mRNA. (E) The expression of GFP mRNA with or without HPLC purification in HEK293T cells were determined by flow cytometry. n = 3 for A, B and n = 4 for E, n refers to the numbers of batches of mRNA transcripts prepared by VSW-3 or T7 RNAP. Data are shown as mean ± SD. Paired t-test was used. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

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  Fig. 4. Incorporation of modified nucleotides for mRNA by VSW-3 or T7 RNAP. (A) Dot-blot analysis of dsRNA signals in GFP mRNA synthesized by T7 or VSW-3 RNAP with modified nucleotides of different proportions (1/4, 2/4, 3/4, 4/4 indicated proportion of modified nucleotides used in IVT). GFP mRNA purchased from Trilink (L-7601) and unmodified GFP mRNA generated by T7 RNAP were served as controls. For the dot blot analysis, 0.1 μg of RNA was loaded for each mRNA. (B–E) The expression of mRNA with various modifications produced by T7 or VSW-3 RNAP in Hela cells was determined by flow cytometry. Flow cytometry charts were shown in B–C. The percentages of GFP-positive cells and MFI were shown in D-E. Data are shown as mean ± SD, n = 3. One-way ANOVA and Dunnett's multiple comparisons test were used. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.