Enhanced high-temperature performance of T7 RNA polymerase by greedy accumulation-directed evolution of thermostability

Abstract

T7 RNA polymerase (T7 RNAP)-catalyzed in vitro transcription (IVT) is the gold standard manufacturing process for large-scale production of therapeutic mRNA molecules. However, the undesired catalytic activity of T7 RNAP concomitantly generates deleterious impurities, such as double-stranded RNAs, that can exacerbate the downstream purification burden and engender safety concerns. The aim of this study was to engineer T7 RNAP thermostability for high-temperature IVT to reduce the dsRNA. The web server PROSS was utilized to predict thermostable mutation sites from the intermediate and elongation structure of T7 RNAP. Through systematic evaluation of individual mutation sites followed by greedy-accumulation optimization of multi-site combinatorial mutants, we successfully overcame the inherent activity-stability trade-off during the evolution and obtained a thermostable variant, M10 (T_m: 49.5°C), which exhibits robust catalytic activity at elevated temperatures and significantly reduced dsRNA byproduct formation. Structural analysis using homology modelling and molecular dynamics (MD) simulation revealed that the accumulated mutations increased the local rigidity of T7 RNAP with a compacted conformation, enhanced the helical propensity, and allowed the formation of new salt bridges. The enhanced mutant has the potential to act as an effective biocatalyst for high-temperature IVT, adding in high-quality mRNA production, which is a prerequisite for optimizing the downstream purification processes and improving the clinical viability of such therapeutic agents.

Graphical Abstract

The PROSS sever was used to predict thermostable mutations in T7 RNAP. A greedy-accumulation approach led to the creation of multi-site combinatorial mutants of T7 RNAP, exhibiting robust catalytic activity at higher temperatures and significantly lower levels of dsRNA byproducts

Keywords

T7 RNAP / In vitro transcription / Thermostability / mRNA therapeutics / dsRNA

References

[1.] BhatB, KarveS, AndersonDG. mRNA therapeutics: beyond vaccine applications. Trends Mol Med, 2021, 279923-4.

[2.] McNamaraJO, GiangrandePH. Toward the full potential of mRNA therapeutics. Mol Ther, 2024, 3261600-1.

[3.] ParhizH, Atochina-VassermanEN, WeissmanD. mRNA-based therapeutics: looking beyond COVID-19 vaccines. Lancet, 2024, 403104321192-204.

[4.] ShenG, LiuJ, YangH, XieN, YangY. mRNA therapies: pioneering a new era in rare genetic disease treatment. J Controlled Release, 2024, 369: 696-721.

[5.] CondeJ, LangerR, RueffJ. mRNA therapy at the convergence of genetics and nanomedicine. Nat Nanotechnol, 2023, 186537-40.

[6.] LenkR, KleindienstW, SzabóGT, BaiersdörferM, BorosG, KellerJM, MahinyAJ, VlatkovicI. Understanding the impact of in vitro transcription byproducts and contaminants. Front Mol Biosci, 2024, 11: 1426129.

[7.] KwonH, KimM, SeoY, MoonYS, LeeHJ, LeeK, LeeH. Emergence of synthetic mRNA: in vitro synthesis of mRNA and its applications in regenerative medicine. Biomaterials, 2018, 156: 172-93.

[8.] RosaSS, PrazeresDMF, AzevedoAM, MarquesMPC. mRNA vaccines manufacturing: challenges and bottlenecks. Vaccine, 2021, 39162190-200.

[9.] WebbC, IpS, BathulaNV, PopovaP, SorianoSKV, LyHH, EryilmazB, Nguyen HuuVA, BroadheadR, RabelM, VillamagnaI, AbrahamS, RaeesiV, ThomasA, ClarkeS, RamsayEC, PerrieY, BlakneyAK. Current status and future perspectives on MRNA drug manufacturing. Mol Pharm, 2022, 1941047-58.

[10.] NanceKD, MeierJL. Modifications in an emergency: the role of N1-Methylpseudouridine in COVID-19 vaccines. ACS Cent Sci, 2021, 75748-56.

[11.] WarneN, RueschM, SiwikP, MensahP, LudwigJ, HripcsakM, GodavartiR, PrigodichA, DolstenM. Delivering 3 billion doses of Comirnaty in 2021. Nat Biotechnol, 2023, 412183-8

[12.] SzabóGT, MahinyAJ, VlatkovicI. COVID-19 mRNA vaccines: platforms and current developments. Mol Ther, 2022, 3051850-68.

[13.] BorkotokyS, MuraliA. The highly efficient T7 RNA polymerase: A Wonder macromolecule in biological realm. Int J Biol Macromol, 2018, 118: 49-56.

[14.] ShisDL, BennettMR. Synthetic biology: the many facets of T7 RNA polymerase. Mol Syst Biol, 2014, 107745.

[15.] CamperiJ, LippoldS, AyalewL, RoperB, ShaoS, FreundE, NissenbaumA, GalanC, CaoQ, YangF, YuC, GuilbaudA. Comprehensive impurity profiling of mRNA: evaluating current technologies and advanced analytical techniques. Anal Chem, 2024, 9693886-97.

[16.] YuM, LevineSJ. Toll-like receptor, RIG-I-like receptors and the NLRP3 inflammasome: key modulators of innate immune responses to double-stranded RNA viruses. Cytokine Growth Factor Rev, 2011, 22263-72.

[17.] CottrellKA, AndrewsRJ, BassBL. The competitive landscape of the DsRNA world. Mol Cell, 2024, 841107-19.

[18.] KarikóK, MuramatsuH, LudwigJ, WeissmanD. Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic Acids Res, 2011, 3921e142.

[19.] CamperiJ, RoperB, FreundE, LeylekR, NissenbaumA, GalanC, CaothienR, HuZ, KoP, LeeA, ChatlaK, AyalewL, YangF, LippoldS, GuilbaudA. Exploring the impact of in Vitro-Transcribed mRNA impurities on cellular responses. Anal Chem, 2024, 964417789-99.

[20.] QuJ, NairA, MuirGW, LovedayKA, YangZ, NourafkanE, WelbourneEN, MaamraM, DickmanMJ, KisZ. Quality by design for mRNA platform purification based on continuous oligo-dT chromatography. Mol Ther Nucleic Acids, 2024, 354102333.

[21.] FengX, SuZ, ChengY, MaG, ZhangS. Messenger RNA chromatographic purification: advances and challenges. J Chromatogr A, 2023, 1707: 464321.

[22.] Zhang J, Liu Y, Li C, Xiao Q, Zhang D, Chen Y, Rosenecker J, Ding X, Guan S. Recent advances and innovations in the Preparation and purification of in Vitro-Transcribed-mRNA-Based molecules. Pharmaceutics. 2023;15(9).

[23.] WuMZ, AsaharaH, TzertzinisG, RoyB. Synthesis of low immunogenicity RNA with high-temperature in vitro transcription. RNA, 2020, 263345-60.

[24.] DousisA, RavichandranK, HobertEM, MooreMJ, RabideauAE. An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts. Nat Biotechnol, 2023, 414560-8.

[25.] MillerM, AlvizoO, BaskervilleS, ChintalaA, ChngC, DassieJ, DorigattiJ, HuismanG, JenneS, KadamS, LeatherburyN, LutzS, MayoM, MukherjeeA, SeroA, SundsethS, PenfieldJ, RigginsJ, ZhangX. An engineered T7 RNA polymerase for efficient co-transcriptional capping with reduced DsRNA byproducts in mRNA synthesis. Faraday Discuss, 2024, 2520431-49.

[26.] HeW, ZhangX, ZouY, LiJ, ChangL, HeYC, JinQ, YeJ. Effective synthesis of circrna via a thermostable T7 RNA polymerase variant as the catalyst. Front Bioeng Biotechnol, 2024, 12: 1356354.

[27.] GoldenzweigA, GoldsmithM, HillSE, GertmanO, LaurinoP, AshaniY, DymO, UngerT, AlbeckS, PriluskyJ, LiebermanRL, AharoniA, SilmanI, SussmanJL, TawfikDS, FleishmanSJ. Automated Structure- and Sequence-Based design of proteins for high bacterial expression and stability. Mol Cell, 2016, 632337-46.

[28.] Van Der SpoelD, LindahlE, HessB, GroenhofG, MarkAE, BerendsenHJ. GROMACS: fast, flexible, and free. J Comput Chem, 2005, 26161701-18.

[29.] HuangJ, RauscherS, NawrockiG, RanT, FeigM, de GrootBL, GrubmüllerH, MacKerellADJr. CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nat Methods, 2017, 14171-3.

[30.] HouQ, RoomanM, PucciF. Enzyme stability-Activity Trade-Off: new insights from protein stability weaknesses and evolutionary conservation. J Chem Theory Comput, 2023, 19123664-71.

[31.] TeuflM, ZajcCU, TraxlmayrMW. Engineering strategies to overcome the Stability–Function Trade-Off in proteins. ACS Synth Biol, 2022, 1131030-9.

[32.] KyngäsJ, ValjakkaJ. Unreliability of the Chou-Fasman parameters in predicting protein secondary structure. Protein Eng, 1998, 115345-8.

[33.] GongH, FreedKF. Electrostatic solvation energy for two oppositely charged ions in a solvated protein system: salt bridges can stabilize proteins. Biophys J, 2010, 983470-7.

[34.] ChanCH, YuTH, WongKB. Stabilizing salt-bridge enhances protein thermostability by reducing the heat capacity change of unfolding. PLoS ONE, 2011, 66e21624.

[35.] Ban X, Wu J, Kaustubh B, Lahiri P, Dhoble A.S., Gu Z, Li C, Cheng L, Hong Y, Tong Y, Li Z. Additional salt bridges improve the thermostability of 1,4-α-glucan branching enzyme. Food Chem. 2020;316:p126348.

[36.] CharbonneauDM, BeauregardM. Role of key salt bridges in thermostability of G. thermodenitrificans EstGtA2: distinctive patterns within the new bacterial lipolytic enzyme family XV. PLoS ONE, 2013, 810e76675.

[37.] McManusTJ, WellsSA, WalkerAB. Salt Bridge impact on global rigidity and thermostability in thermophilic citrate synthase. Phys Biol, 2019, 171016002.

Funding

Social Development Key Project of Jiangsu Province(BE2023682)