Fig. 3

PERSIST-seq summary and representative ribosome load findings. a Schematic representation of the mRNA optimization process. 5′ and 3′ UTRs sourced from literature and rational design were merged with Eterna and algorithmically created coding sequences. All sequences underwent simultaneous experimental evaluation for in-solution and in-cell stability, as well as ribosome load. Unique 6–9 nt barcodes in the 3′ UTR of the mRNA design enabled tag counting via short-read sequencing. b Experimental layout for assessing in-solution and in-cell stability and ribosome load concurrently. mRNAs were in vitro transcribed, 5′ capped, and polyadenylated in a pooled format prior to HEK293T cell transfection or in-solution degradation exposure. Cells were then collected for sucrose gradient fractionation or in-cell degradation examination. c Polysome trace from a 233-mRNA pool transfected into HEK293T cells. d 5′ UTR variations exhibit greater variability in average ribosome load per construct, as determined by polysome sequencing. The ribosome load formula is provided. Box hinges display 25% quantile, median, and 75% quantile from left to right, while whiskers indicate lower or upper hinge ± 1.5 × interquartile range. e Heatmaps of polysome profiles for top, middle, and bottom five mRNA designs (based on ribosome load) from each design category. f SARS-CoV-2 5′ UTR secondary structure model, with highlighted mutations and substitutions. g Heatmaps of SARS-CoV-2 5′ UTR variant polysome profiles, sorted by ribosome load. Reprinted from [87] with permission from Springer Nature