Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

Figures

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  Figure 1.. PERSIST-seq overview and illustrative ribosome load insights. (A) Overview of the mRNA optimization workflow. Literature mined and rationally designed 5’ and 3’ UTRs were combined with Eterna and algorithmically designed coding sequences. All sequences were then experimentally tested in parallel for in-solution and in-cell stability as well as ribosome load. The mRNA design included unique, 6–9 nt barcodes in the 3’ UTR for tag counting by short-read sequencing. (B) Experimental design for testing in-solution and in-cell stability and ribosome load in parallel. mRNAs were in vitro transcribed, 5’ capped, and polyadenylated in a pooled format before transfection into HEK293T cells or being subjected to in-solution degradation. Transfected cells were then harvested for sucrose gradient fractionation or in-cell degradation analysis. (C) Polysome trace from transfected HEK293T cells with 233-mRNA pool. (D) 5’ UTR variants display a higher variance in mean ribosome load per construct as determined from polysome sequencing. The formula for ribosome load is given. (E) Heatmaps from polysome profiles of mRNA designs selected from the top, middle, and bottom five mRNAs (by ribosome load)…

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  Figure 2.. In-cell RNA stability drives downstream protein expression levels. (A) In-cell half-life of each mRNA design in HEK293T cells. (B) Higher polysome load correlates with decreased in-cell half-life. Correlation between in-cell half-life and mean ribosome load across the entire profile (left), monosome-to-free subunit ratio (center), or polysome-to-monosome ratio (right). (C) In-cell half-life and mean ribosome load for individual mRNA designs with varying UTRs. (D) Kinetic model for predicting protein expression from mRNA half-life and ribosome load. P(t) is protein quantity at time t; m0 is the mass of mRNA present at t=0; l is mRNA length; kt is translation rate; and km and kp are rates of mRNA and protein decay, respectively. (E) Protein expression predicted using the kinetic model in (D) on the basis of mRNA half-life and ribosome load. Top: predicted protein expression of each UTR variant; note closer similarity to in-cell half-life data than to ribosome load in (C). Bottom: predicted protein expression normalized by mRNA length (corresponding to transfecting equal masses of each mRNA). (F) Correlation of predicted protein expression and Nluc/Fluc activity in HEK293T cells.…

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  Figure 3.. High-throughput in-line hydrolysis uncovers principles of in-solution RNA degradation. (A) Eterna participants were asked to design 68-nucleotide RNA fragments maximizing sequence and structure diversity. 3030 constructs were characterized and probed using high-throughput in-line degradation (In-line-seq). (B) Nucleotide-resolution degradation of 2165 68-nt RNA sequences (filtered for signal quality), probed by In-line-seq, sorted by hierarchical clustering on degradation profiles. (C) Sequences span a diverse set of secondary structure motifs, revealing patterns in degradation based on both sequence (i.e., linkages ending at 3’ uridine are particularly reactive) and structure (symmetric internal loops, circled, have suppressed hydrolytic degradation compared to asymmetric internal loops). (D) The ridge regression model “DegScore” was trained to predict per-nucleotide degradation from sequence and loop assignment information. Coefficients with the largest magnitude corresponded to sequence identity immediately after the link, with U being most disfavored. (E) DegScore showed improved predictive power on mRNAs over two other metrics previously posited to predict RNA stability. (F)…

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  Figure 4.. Integration of 5’/3’ UTRs, structure-optimized CDSs, and pseudouridine (ψ) together enhance mRNA stability and translational output. (A) CDS and 5’/3’UTR combinations differentially impact protein synthesis. Six mRNA constructs were in vitro synthesized and luciferase activity was measured 6 or 24 hrs post-transfection. Inclusion of ψ was tested on two selected constructs. (B) Workflow for different approaches to design the CDS variants tested in (C). (C) Variations in CDS design facilitates high in-solution stability and differential protein expression. In vitro transcribed mRNAs (24 in total) were subjected to in-solution degradation or transfected into HEK293T cells for 6 and 24 hrs. In-solution half-lives and luciferase activity are normalized to the Nluc START reference construct. Predicted secondary structures are shown for select constructs with colors indicating DegScore at each nucleotide. Designs derived from LinearDesign solutions are marked with a purple triangle. (D) Predicted secondary structure overview of Ribotree_LinearDesign_degscoreall_1. Zoomed boxes indicate sequence optimizations and subsequent structural changes made by DegScore to the reference…