Kathrin Leppek, Gun Woo Byeon, Wipapat Kladwang, Hannah K. Wayment-Steele, Craig H. Kerr et al.
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.
Kathrin Leppek, Gun Woo Byeon, Wipapat Kladwang, Hannah K. Wayment-Steele, Craig H. Kerr, Adele F. Xu, Do Soon Kim, Ved V. Topkar, Christian Choe, Daphna Rothschild, Gerald C. Tiu, Roger Wellington-Oguri, Kotaro Fujii, Eesha Sharma, Andrew M. Watkins, John J. Nicol, Jonathan Romano, Bojan Tunguz, Fernando Diaz, Hui Cai, Pengbo Guo, Jiewei Wu, Fanyu Meng, Shuai Shi, Eterna Participants, Philip R. Dormitzer, Alicia Solórzano, Maria Barna, Rhiju Das. Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics. 2022-03-22. https://doi.org/10.1038/s41467-022-28776-w
A 233-member library of full-length, in-vitro-transcribed reporter mRNAs with unique 3′-UTR barcodes was transfected into HEK293T cells and separated by sucrose-gradient polysome profiling. Barcode sequencing across the fractions quantifies construct-specific translation/ribosome loading.
The 233-member synthetic mRNA pool was subjected to accelerated in-solution degradation and sampled over ten time points; intact full-length molecules were quantified by barcode sequencing. Exponential fits provide construct-specific degradation coefficients and half-lives.
The same 233 full-length barcoded reporter mRNAs were transfected into HEK293T cells, harvested over the in-cell time course, and quantified by full-length RT-PCR followed by barcode sequencing. The resulting normalized abundance trajectories quantify construct-specific mRNA decay and half-life.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 12 files (ZIP)barcode_reference_233pool.fabarcode_reference_233pool.txtGSE173083_family.soft.gzGSE173083_Table_S1_Attributes_for_pooled_233_sequences.xlsxin_solution_raw_all_counts.tsvin_solution_raw_samples.tsvpersist_analysis_constructs.tsvpersist_analysis_joined_counts.txtpersist_analysis_persist_p.Rpersist_analysis_README.mdpersist_analysis_samples.csvSupplementary_Data_7_Primers_and_construct_layouts.xlsx