Figure Legends
Figure 1 - mRNA structures were engineered to increase product stability in E. coli host-cells by A) including ‘scaffold’ tRNA-Lysine motifs at both the 5’ and 3’ termini, B)incorporating ribozyme sequences to promote self-circularisation, andC) inserting component parts of the tRNA-Lysine motif at the 5’ and 3’ termini to facilitate formation of pseudo-circular molecules. Designed elements were synthesized, individually inserted into a GFP-expression plasmid and evaluated in 2.5 h production processes(D) . Data are expressed as a fold-change of the production exhibited by the unengineered control system. Values represent the mean + SD  of three independent experiments (n  = 3, each performed in triplicate). UTR: Untranslated region; IRES: Internal ribosome entry site; ORF: Open reading frame; GFP: Green Fluorescent Protein.
Figure 2 - mRNA manufacturing platform components were sequentially optimised by evaluating the function of A)engineered host cell chassis, B) synthetic DNA expression vectors, and C) designed cell culture media formulations. The relative performance of engineered systems was evaluated in 2.5 h production processes. The additive impact of each engineering step on overall product yield is shown in D . Data are expressed as a fold-change of the production achieved using standard control components. Values represent the mean + SD  of three independent experiments (n  = 3, each performed in triplicate). In all panels, the expressed product is SelfCirc-GFP (see Fig. 1); the effect of whole system engineering on manufacture of TermtRNA-GFP is shown in Supplementary figure 2.
Figure 3 - A) Capillary electropherograms of total RNA isolated from GFP-mRNA biomanufacturing systems comprising either i) standard control components, or ii) an optimal combination of engineered mRNA construct, DNA expression plasmid, cell host and media formulation (Engineered system, see Fig. 2). B) Gel electrophoresis analysis of total RNA isolated from Engineered systems producing Green fluorescent protein (GFP), SARS-COV-2 Spike and Cypridina Luciferase mRNA molecules. Full-length product mRNA molecules are highlighted by red arrows; molecule sizes are enlarged due to the presence of IRES and Intron elements in SelfCirc-mRNA (see Fig. 1). C) Comparison of relative GFP-mRNA yields obtained from Standard control and Engineered systems, quantified before (intracellular) and after purification using oligo-dT magnetic beads. Intracellular yields are expressed as a fold-change of the product yield obtained using the Standard control system. Values represent the mean + SD  of three independent experiments (n  = 3, each performed in triplicate).
Figure 4 - SelfCirc-Spike and TermtRNA-Spike were produced in biomanufacturing systems comprising an optimal combination of engineered host cell, DNA plasmid and cell culture media (see Fig 2). Growth of non-producer (uninduced) and producer cells was measured at 1 h intervals during 6 h production processes (A) , to calculate integral cell concentration (ICC) and cell specific growth rate (u) values (B) . Total RNA (C) and product mRNA(E) yields were also measured at 1 h intervals, and the relative proportion of host cell RNA comprising Spike-mRNA molecules was quantified (D) . Total RNA samples from each measured timepoint were analysed by capillary gel electrophoresis; dashed red boxes on capillary electropherograms highlight SelfCirc-Spike (F) and TermtRNA-Spike (G) product accumulating over time. In A-C, values represent the mean ± SD of three independent experiments; D-G show data from a single representative capillary gel electrophoresis timecourse analysis.
Figure 5 . A) Simplified process flow diagram for large-scale and small-scale in vivo mRNA production. B)Typical chromatogram from oligo-d(T) affinity chromatography purification of product mRNA manufactured in E. coli .C-D) Capillary electropherograms showing purification of TermtRNA-GFP (C) and SelfCirc-GFP (D) products using oligo-d(T) affinity chromatography. E-F) mRNA products manufactured in E. coli were purified and transfected into Human Embryonic Kidney cells. 24 hr posttransfection, GFP protein expression was measured by fluorescent cell imaging (E) and fluorescent plate reader analysis (F). Values in F represent the mean + SD  of three independent experiments (n  = 3, each performed in triplicate).