biostudies-literatureLeppek KNICHD NIH HHSNCI NIH HHSNIGMS NIH HHS1536https://www.ebi.ac.uk/biostudies/studies/S-EPMC8940940biostudies-literatureUnknown13(1)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 an 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 highly structured "superfolder" mRNAs can be designed to improve both stability and expression with further enhancement 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.Nature communicationsCombinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics.PMC8940940F30 HD100123R35 GM122579R01 HD086634R21 CA219847Wu JParticipants EByeon GWFujii KDas RXu AFShi SBarna MTopkar VVKerr CHLeppek KCai HGuo PWayment-Steele HKTunguz BKim DSSharma EKladwang WRomano JSolorzano AWellington-Oguri RTiu GCWatkins AMMeng FRothschild DDiaz FChoe CNicol JJDormitzer PRfalseCombinatorial 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 an 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 highly structured "superfolder" mRNAs can be designed to improve both stability and expression with further enhancement 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.2022-01-01T00:00:00Z2022 Mar2026-06-18T08:47:04.657Z2025-04-06T21:51:06.408ZS-EPMC89409403531832410.1038/s41467-022-28776-w