The RNA Landscape of In Vivo-Assembled MS2 Virus-Like Particles as mRNA Carriers Reveals RNA Contamination from Host Viruses

Figures

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  Figure 1. Schematic illustration of mRNA encapsulation into MS2 VLPs in yeast. MS2 CPs and EGFP with varying numbers of pac sites were coexpressed under the control of GAL1 and GAL10 promoters, respectively. MS2 CPs coassembled with EGFP mRNA and formed VLPs, with the encapsulation efficiency being regulated by the copy number of pac sites. The VLPs were isolated, and the landscapes of RNA inside were analyzed. Created in BioRender. Ziyan, H. (2025) https://BioRender.com/l93l587.

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  Figure 2. Characterization of purified MS2 VLPs coassembled with EGFP mRNA carrying varying numbers of pac sites. (a) SDS-PAGE (left) and TEM (right) characterization of the purified VLPs. Scale bars: 50 nm. (b) Agarose gel electrophoresis of PCR products. Total RNA was extracted from the VLPs and reverse-transcribed (RT) to cDNA. Then, PCR was conducted with cDNA as templates to test the presence of EGFP (720 bp) and MS2 CP (390 bp) mRNA in the VLPs. The RNA-marked lanes represent no-RT control PCR performed on the same total RNA using the same primers and polymerase to verify the absence of DNA contamination.

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  Figure 3. Quantitative PCR analysis of target mRNA. (a) Amplification plots of qPCR showing the cycle-by-cycle accumulation of fluorescence, indicative of the amplification of EGFP and MS2 CP mRNA within samples under gradient dilution, 10–1 to 10–5. (b) Standard curves of the cycle threshold (Ct) value versus the log10 of the initial DNA copy number of the EGFP or MS2 CP gene, demonstrating linear amplification across the range of template concentrations. The high R2 values indicate a good correlation between the logarithm of the sample concentration and the Ct value. Both EGFP and MS2 CP amplification efficiencies (E) fell within the acceptable range of 90–110%, confirming the reliability and accuracy of the qPCR data.32 (c) mRNA copies per 1000 VLPs for EGFP and MS2 CP. Data are derived from three independent qPCR experiments and are presented as mean ± SD. (d) Correlation between the RNA copy number per 1000 VLPs and the designed pac number.

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  Figure 4. Transcriptome sequencing analysis of the total RNA extracted from the engineered MS2 VLPs. (a) The reads categorization of various RNA types encapsulated within E-0pac, E-1pac, and E-3pac VLPs. Reference RNA species included EGFP, MS2 CP, YPH499 genome, and pESC-URA plasmid sequences. “Others” represents sequences that were not mapped to the aforementioned references. Data are presented as mean ± SD from three independent experimental replicates. (b) The histogram presents a further classification of the unmapped reads based on the NCBI nucleotide database. The three bars corresponding to each type of VLP represent transcriptome analyses from three different samples.

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  Figure 5. Characterization of the purified E-3pac VLPs after further separation. (a) SEC of the purified E-3pac VLPs. (b, c) TEM images (b) and size distributions (c) of the samples from peaks A and B. Peaks A and B contained VLPs with mean diameters of ca. 38.1 and 26.7 nm, respectively. Scale bars, 50 nm. (d) SDS-PAGE of the samples collected from peaks A and B.