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

Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop a new RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that “superfolder” mRNAs can be designed to improve both stability and expression that are further enhanced through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines. Overall design: For polysome profiling experiments, 16 polysome fractions were collected and sequenced. For RNA degradation experiments in cells, 5 timepoints with 3 replicates each were collected. For RNA degradation experiments in solution, 10 timepoints were collected. For directed evolution experiments, two RNA pools were subject to five cycles of selection. For both pools, the input and selection results of cycles 0, 1, and 5 were sequenced. The selected results of cycles 2, 3, 4 were also sequenced. For RNA chemical mapping experiments, Yellowstone, Lineardesign constructs were probed with 1M7, DMSO, DMS, and NM (water). For P4P6 control, treatment was done with DMS and NM (water). For in-line-seq degradation experiments, 45-50 pmol of RNA was subject to four conditions: 1) 50 mM Na-CHES buffer (pH 10.0) at room temperature without added MgCl2 ; 2) 50 mM Na-CHES buffer (pH 10.0) at room temperature with 10 mM MgCl2 ; 3) phosphate buffered saline (PBS, pH7.2; Thermo Fisher Scientific-Gibco 20012027) at 50°C without added MgCl2 ; and 4) PBS (pH7.2) at 50°C with 10 mM MgCl2. For degradation reactions containing MgCl2, RNA was collected at 0 and 24 hour time points. For reactions without MgCl2, timepoints were collected at 0 and 7 days. In-line-seq RNA libraries were also probed for structure with the addition of 1M7 or no 1M7 (negative control)