Fig. 1: Construction and delivery mechanisms of the Gemini 1.0 vector. | Nature Communications

Fig. 1: Construction and delivery mechanisms of the Gemini 1.0 vector.

From: A binary self-amplifying expression platform enabling lipid nanoparticle-free vaccines and nanomedicines

Fig. 1

A Genomic map of the Gemini 1.0 vector. The vector contains a binary promoter (CMV and T7), replicon protein genes (NSP1-4), a 26S sub-genomic promoter from the Venezuelan equine encephalitis (VEE) virus, and resistance genes for puromycin (PuroR) and ampicillin (AmpR) for mammalian and bacterial cell culture, respectively. (Created in BioRender. Fan J. (2025) https://BioRender.com/hfk8ebr). B Delivery mechanisms of Gemini as saDNA or saRNA. (Left panel) saDNA is delivered directly into the cell; it enters the nucleus for transcription then the resulting mRNA is transported to the cytoplasm for translation using the endogenous machinery to express its RNA dependent RNA polymerase (RdRp). (Right panel) saRNA is delivered into the cytoplasm for direct translation of its RdRp by the endogenous machinery. The RdRp then transcribes the payload mRNA, allowing for production of the payload protein. saRNA is transcribed in vitro via the T7 promoter with assistance from T7 polymerase. Also, 5’ capping is performed in vitro, before mRNA delivery into the cytoplasm for direct translation of RdRp and subsequent expression of payload mRNA. (Created in BioRender. Fan J. (2025) https://BioRender.com/hfk8ebr). C, D Establishing Negative-strand RNA synthesis is present post-transfection with naked  saRNA-eGFP and naked saDNA-eGFP. C Detection strategy: First-strand synthesis is conducted using a NSP4 region specific primer (purple) with a random nucleotide tag (red) to produce positive strand (+) cDNA. The negative (-) RNA strand is then synthesized as cDNA using primers specific for the eGFP region (green) and the random nucleotide tag, producing a band of about 1.9 kb. A subsequent nested PCR (primers: dark green and fuchsia) then produces a band of 1.4 kb. (Created in BioRender. Fan J. (2025) https://BioRender.com/8t59tko). D Expression of both Gemini saDNA and saRNA are demonstrated in vitro through PCR results shown on agarose gel: Total RNA was collected from HEK293 cells transfected with naked saRNA and saDNA expressing eGFP. Transfected HEK293 cells were grown in the absence of subsequent drug selection. Results of the first and nested PCRs are shown when the forward primer was used and not used (i.e., controls). The 1Kb Plus DNA Ladder (Froggobio) in lane 1 indicates the position and sizes of the molecular weight markers in base pairs (bp).

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