Engineering of enzymatic modules for mRNA manufacturing: Advances in catalytic regulation and process integration

doi:10.1016/S1872-2067(25)64860-1

Chinese Journal of Catalysis ›› 2026, Vol. 80: 92-112.DOI: 10.1016/S1872-2067(25)64860-1

• Reviews • Previous Articles Next Articles

Engineering of enzymatic modules for mRNA manufacturing: Advances in catalytic regulation and process integration

Shiyi Che^a^,^b, Zhengjun Li^b, Zhiguo Su^b, Zhikao Li^a, Aibing Yu^a, Minsu Liu^a^,^c^,^*(), Songping Zhang^b^,^*()

^aARC Research Hub for Smart Process Design and Control, Department of Chemical and Biological Engineering, Monash University, Clayton, Victoria 3800, Australia
^bState Key Laboratory of Biopharmaceutical Preparation and Delivery, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China
^cDepartment of Materials Science and Engineering, Monash University, Clayton, Victoria 3800, Australia

Received:2025-06-09 Accepted:2025-09-06 Online:2026-01-18 Published:2026-01-05
Contact: Minsu Liu, Songping Zhang
About author:Minsu Liu (Monash University) is a senior research fellow at Monash Suzhou Research Institute and a senior lecturer at Department of Materials Science and Engineering, Monash University. He has obtained his B.E. (Hon. 1) and Ph.D. from the University of New South Wales (Australia) and Monash University (Australia), respectively. Before joining Monash, Dr. Liu worked as postdoctoral fellow at Monash University (2017‒2018) and Tsinghua University (2018‒2020). Dr. Minsu Liu is expertise in the research of nanofabrication, 2D materials, self-assembly, and thermal management. He has published more than 50 peer-reviewed journal articles with more than 2000 citations, and applied/granted more than 50 patents.
Songping Zhang (Institute of Process Engineering, Chinese Academy of Sciences) received her B.S. in 1998 and Ph.D degree in 2002 from Tianjin University, China. From 2002‒2004, she did postdoctoral research at Lund University, Sweden. Afterward, she joined Institute of Process Engineering in Jan. 2005. Prof. Zhang’ research direction focusses on biomedicine engineering, specializing in the synthesis, assembly, purification, and formulation of biomacromolecules including mRNA, proteins, recombinant virus like particles vaccine antigens, et al. She has published over 150 research papers and holds more than 40 invention patents, with multiple achievements succeed in industrial applications. She serves as an editorial board member for J. Chromatogr. A and Biochem. Engin. J.
Supported by:
National Natural Science Foundation of China(22478401);Strategic Priority Research Program of the Chinese Academy of Sciences(XDB1250000);Australian Research Council (ARC)(IH230100010)

Abstract

Abstract:

The clinical efficacy of mRNA-based therapeutics is critically dependent on the structural integrity of the mRNA molecule, which in turn is governed by the efficiency and robustness of its manufacturing process. Unlike conventional small-molecule synthesis, mRNA manufacturing relies on complex enzymatic cascades involving biomacromolecules with dynamic conformations as templates, intermediates, and catalysts. Key enzymatic modules, including plasmid linearization for DNA template preparation (Module 1), in vitro transcription (IVT) synthesis (Module 2), capping modification (Module 3) of mRNA, and different nucleases-aided removal of impurities (Module 4), are highly interdependent, each with specific catalytic enzymes and auxiliary cofactors. These modules present major engineering challenges of low efficiency and lack of modular compatibility across the multi-step enzymatic processes. Moreover, traditional approaches such as multienzyme immobilization or compartmentalization often fail to meet the demands of high-throughput, continuous and scalable manufacturing. This review systematically summarizes recent advances in the engineering of enzymatic modules for mRNA manufacturing, emphasizing challenges in catalytic regulation, module integration and process intensification. The potential strategies for improving reaction compatibility and enabling process integration and intensification are discussed, providing insights into future directions for engineering mRNA synthesis at scale.

Key words: mRNA manufacturing, Enzymatic modules, Catalytic regulation, Process integration, Multienzyme reaction

Shiyi Che, Zhengjun Li, Zhiguo Su, Zhikao Li, Aibing Yu, Minsu Liu, Songping Zhang. Engineering of enzymatic modules for mRNA manufacturing: Advances in catalytic regulation and process integration[J]. Chinese Journal of Catalysis, 2026, 80: 92-112.

×
share this article

Add to citation manager EndNote|Ris|BibTeX

URL: https://www.cjcatal.com/EN/10.1016/S1872-2067(25)64860-1

https://www.cjcatal.com/EN/Y2026/V80/I1/92

Figures/Tables 13

Fig. 1. Flow chart for manufacturing of capped mRNA from plasmid preparation to enzymatic capping, which includes four typical catalytic reaction modules. Module 1: Circular plasmid DNA is linearized by the restriction endonuclease (REase). Module 2: The linearized plasmid DNA serves as the template for IVT synthesis of mRNA catalyzed by T7 RNAP along with PPase. Module 3: Enzymatic capping of mRNA by vaccinia capping enzyme (VCE) and 2’-O-methyltransferase (2’-O-MTase) to generate the cap-1 mRNA. Module 4: A panel of nucleases mainly including RNase A, DNase I, RNase III, and RNase R, which are employed to degrade the residual RNA in the plasmid, residual plasmid DNA and dsRNA in the IVT mRNA, linear precursor RNA impurities in the circular RNA, respectively. Between each module, chromatographic processes are integrated to ensure intermediate and final product purity.

Fig. 2. REases for circular plasmid linearization. Left: Different topological forms of plasmid. Right: Two main types of Type II REase cleavages.

Fig. 3. IVT reaction catalyzed by T7 RNAP and PPase. (A) Mechanism of IVT reaction for mRNA synthesis: T7 RNAP catalysis, Mg2PPi byproduct formation and PPase catalysis. Conformational changes of initiation phase (PDB: 1QLN) (B) and elongation phase (PDB: 1H38) (C). (D) NTP insertion. Reprinted with permission from Ref. [37]. Copyright 2009, Elsevier. (E) RNA byproducts. (E-1) Incomplete RNA transcripts. (E-2) Intramolecular (cis) and intermolecular (trans) 3’extension. (E-3) Antisense RNA by promoter-less DNA. (E-4) Non-covalent mRNA aggregates.

Fig. 4. Enzyme engineering and co-immobilization of RNAP. (A) Thermophilic RNAP (G788A). (B) mRNA synthesis by psychrophilic Syn5 RNAP and VSW-3 RNAP. Reprinted with permission from Ref. [54]. Copyright 2022, Taylor & Francis. (C) RNAP for conformation transition (G47A and an extra C-terminal 884G). Reprinted with permission from Ref. [58]. Copyright 2023, Springer Nature. (D) Immobilized strategy for simultaneous tethering T7 RNAP and DNA template. (D-1) Tethering Strep-tag T7 RNAP and 5’-biotin nontemplate strand of DNA to Strep-TactinXT-coated magnetic beads. Reprinted with permission from Ref. [60]. Copyright 2021, Elsevier. (D-2) Tethering 5’-biotin template strand of DNA to streptavidin magnetic beads with promoter on the nontemplate strand linked HaloTag-T7 RNAP. Reprinted with permission from Ref. [61]. Copyright 2024, Oxford University Press.

Table 1 In-process regulation strategies in the IVT module.

Regulation		Impact	Condition	Ref.
Enzyme engineering	thermophilic RNAP	reduced 3’extension dsRNA	high-temperature	[50]
	psychrophilic RNAP	reduced 3’extension dsRNA	low-temperature	[53,54]
	P266L mutant	reduced abortive RNA	—	[56]
	S43Y mutant G47A + 884G mutant	reduced 3’extension dsRNA	—	[57,58]
	G47W mutant	reduced antisense dsRNA	—	[59]
Co-immobilization	DNA was tethered with T7 RNAP to beads	reduced 3’extension dsRNA/Higher yield	high-salt	[60,61]
Using modified NTP	Ψ, m¹Ψ, m⁵C, 5moU	reduced antisense dsRNA	—	[44,69,70]
Using modified NTP	increased ARCA/ m¹Ψ	reduced immunogenicity	—	[75]
Optimizing RNA sequence	-4 gap on the non-template strand of the DNA promoter	reduced 3’extension dsRNA/Higher yield	high-salt	[76]
Optimizing RNA sequence	additional AT-rich sequences at T7 promoter downstream	reduced abortive RNA and dsRNA/Higher yield	high-temperature	[77]
Reaction process regulation	fed-batch strategy	higher yield	—	[78,79]
	low ratio of Mg²⁺/NTP	reduced antisense dsRNA	—	[80]
	low UTP concentrations	reduced 3’extension dsRNA	—	[42,74]
	addition of urea or formamide	reduced 3’extension dsRNA	—	[81,82]

Fig. 5. Reaction process regulation and model. (A-1) The fed-batch strategy with pH-controlled addition of KOH, NTP and Mg(OAc)2. Reprinted with permission from Ref. [78]. Copyright 1999, American Institute of Chemical Engineers. (A-2) The fed-batch strategy with at-line HPLC-monitored addition of NTPs and Mg2+. Reprinted with permission from Ref. [79]. Copyright 2023, Wiley. (B) Bayesian optimization approach with a robotic liquid handler to enable high-throughput algorithmic optimization. Reprinted with permission from Ref. [94]. Copyright 2024, American Chemical Society. (C) A new IVT reaction model reported by Stover et al. Reprinted with permission from Ref. [95]. Copyright 2024, Wiley.

Fig. 6. Conformation of (A) vaccinia capping enzyme (PDB: 4CKB) and (B) 2’-O-methyltransferase (PDB: 1AV6). (A-1) Domains and mRNA capping pathways in VCE [104]. (A-2) FigureTPase with a closed hydrophilic tunnel formed by eight β-barrels. (A-3) NTase domain with GTP-binding site and OB domain in GTase. (A-4) N7-MTase domain with SAM-binding site. Engineering in-process regulation strategies of immobilization of VCE and 2’-O-MTase (C), characterization of novel FCE and its comparison to VCE at different temperatures (Reprinted with permission from Ref. [107]. Copyright 2023, Cold Spring Harbor Laboratory Press) (D), and VCE catalysis with different GTP analogs: Chemical structure and capping efficiency (E). Reprinted with permission from Ref. [113]. Copyright 2023, Oxford University Press.

Table 2 Summary of nucleases in mRNA manufacturing.

Enzyme	Source	Type	Metal ions	Application
RNase A	Bovine pancreas	Endo-	independent	RNA removal in circular plasmid preparation
DNase I	Bovine pancreas	Endo-	Mg²⁺, Ca²⁺	DNA removal after the IVT reaction
RNase III	E. coli	Endo-	Mg²⁺	dsRNA removal after the IVT reaction
RNase R	E. coli	Exo-	Mg²⁺	linear RNA precursor removal in circRNA purification

Fig. 7. Nucleases conformation and engineering in mRNA manufacturing. (A-1) Interactions between RNase A (PDB: 3DXG) and RNase inhibitor (PDB: 4PEQ). (A-2) Immobilization of RNase A by macroporous monolithic support. (B) Adsorption of DNase I (PDB: 1DNK) by AuNP with a capping agent surface. (C) The homodimeric structure of E. coli RNase Ⅲ (PDB: 7R97). (D) Structure of E. coli RNase R. Reprinted with permission from Ref. [133]. Copyright 2017, Oxford University Press.

Fig. 8. Three strategies for process integration in mRNA manufacturing and their corresponding main characteristics and challenges. Briefly, the first strategy is integration between circular plasmid linearization and IVT modules (Modules 1 and 2), the second type is integration between IVT and capping modules (Modules 2 and 3), and the third type is reaction-separation coupling, which usually requires solid-phase-immobilized mRNA. These three reaction modules involved different reaction types of site cleavage, de novo synthesis and site modification, respectively.

Fig. 9. Integration between IVT and capping modules. (A) Co-transcription with cap analogs. (B) Fusion of T7 RNAP and FCE reported by Robb et al. [124]. (C) Continuous cascade reaction by integrated buffer reported by Nwokeoji et al. Reprinted with permission from Ref. [145]. Copyright 2023, American Chemical Society. (D) Continuous process control by digital twin reported by Helgers et al. Reprinted with permission from Ref. [146]. Copyright 2021, MDPI.

Table 3 General conditions of the enzymatic reactions catalyzed by T7 RNAP, VCE and 2’-O-MTase.

Enzyme	Condition
Enzyme	Temperature	pH	Buffer	Mg²⁺	spermidine	DTT	Na⁺/K⁺/Cl^-
T7 RNAP	37 °C	7.5-8.0	Tris-HCl	10-50 mmol/L	1-2 mmol/L, promote DNA condensation	1-10 mmol/L	inhibitory at concentrations > 0.1 mol/L
VCE	37-45 °C			1 mmol/L	—	1 mmol/L
2’-O-MTase	45 °C			1 mmol/L	—	1 mmol/L

Fig. 10. Reaction-separation coupling for mRNA manufacturing. (A) The combined IVT-solid-phase reaction-separation approach published by Liu et al. Reprinted with permission from Ref. [151]. Copyright 2015, Springer Nature. (B) The chemoenzymatic method published by Thillier et al. Reprinted with permission from Ref. [152]. Copyright 2012, Cold Spring Harbor Laboratory Press. (C) The on‐column capping of poly dT-tethered mRNA proposed by Che et al. Reprinted with permission from Ref. [153]. Copyright 2024, Wiley.

References

[1]	N. Al Fayez, M. S. Nassar, A. A. Alshehri, M. K. Alnefaie, F. A. Almughem, B. Y. Alshehri, A. O. Alawad, E. A. Tawfik, Pharmaceutics, 2023, 15, 1972.
[2]	S. Xu, K. Yang, R. Li, L. Zhang, Int. J. Mol. Sci., 2020, 21, 6582.
[3]	V. P. Chavda, R. Pandya, V. Apostolopoulos, Expert Rev. Vaccines, 2021, 20, 1549-1560.
[4]	N. Pardi, M. J. Hogan, F. W. Porter, D. Weissman, Nat. Rev. Drug Discov., 2018, 17, 261-279.
[5]	A. Hınçer, R. E. Ahan, E. Aras, U. Ö. Ş. Şeker, ACS Synth. Biol., 2023, 12, 2505-2515.
[6]	E. Oude Blenke, E. Örnskov, C. Schöneich, G. A. Nilsson, D. B. Volkin, E. Mastrobattista, Ö. Almarsson, D. J. A. Crommelin, J. Pharm. Sci., 2023, 112, 386-403.
[7]	S. Bancel, W. J. Issa, J. G. Aunins, T. Chakraborty,WO Patent 2014152027A1, 2014.
[8]	X. Feng, Z. Su, Y. Cheng, G. Ma, S. Zhang, J. Chromatogr. A, 2023, 1707, 464321.
[9]	K. J. Prather, S. Sagar, J. Murphy, M. Chartrain, Enzyme Microb. Technol., 2003, 33, 865-883.
[10]	J. Ingels, L. De Cock, R. L. Mayer, P. Devreker, K. Weening, K. Heyns, N. Lootens, S. De Smet, M. Brusseel, S. De Munter, M. Pille, L. Billiet, G. Goetgeluk, S. Bonte, H. Jansen, S. Van Lint, G. Leclercq, T. Taghon, B. Menten, K. Vermaelen, F. Impens, B. Vandekerckhove, Cytotherapy, 2022, 24, 213-222.
[11]	D. Kairuz, N. Samudh, A. Ely, P. Arbuthnot, K. Bloom, Front. Immunol., 2022, 13, 1018961.
[12]	J. M. Henderson, A. Ujita, E. Hill, S. Yousif-Rosales, C. Smith, N. Ko, T. McReynolds, C. R. Cabral, J. R. Escamilla-Powers, M. E. Houston, Curr. Protoc., 2021, 1, e39.
[13]	R. Tenchov, R. Bird, A. E. Curtze, Q. Zhou, ACS Nano, 2021, 15, 16982-17015.
[14]	S. S. Rosa, D. M. F. Prazeres, A. M. Azevedo, M. P. C. Marques, Vaccine, 2021, 39, 2190-2200.
[15]	R. Verbeke, M. J. Hogan, K. Loré, N. Pardi, Immunity, 2022, 55, 1993-2005.
[16]	S. Plotkin, J. M. Robinson, G. Cunningham, R. Iqbal, S. Larsen, Vaccine, 2017, 35, 4064-4071.
[17]	M. Youssef, C. Hitti, J. Puppin Chaves Fulber, A. A. Kamen, Biomolecules, 2023, 13, 1497.
[18]	U. Chheda, S. Pradeepan, E. Esposito, S. Strezsak, O. Fernandez-Delgado, J. Kranz, J. Pharm. Sci., 2024, 113, 377-385.
[19]	S. Daniel, Z. Kis, C. Kontoravdi, N. Shah, Trends Biotechnol., 2022, 40, 1213-1228.
[20]	E. Hajnsdorf, V. R. Kaberdin, Phil. Trans. R. Soc. B, 2018, 373, 20180166.
[21]	Z. Trepotec, J. Geiger, C. Plank, M. K. Aneja, C. Rudolph, RNA, 2019, 25, 507-518.
[22]	C. Y. Li, Z. Liang, Y. Hu, H. Zhang, K. D. Setiasabda, J. Li, S. Ma, X. Xia, Y. Kuang, Mol. Ther. Nucleic Acids, 2022, 30, 300-310.
[23]	R. H. Durland, E. M. Eastman, Adv. Drug Deliv. Rev., 1998, 30, 33-48.
[24]	A. Xenopoulos, P. Pattnaik, Expert Rev. Vaccines, 2014, 13, 1537-1551.
[25]	A. Pingoud, G. G. Wilson, W. Wende, Nucleic Acids Res., 2014, 42, 7489-7527.
[26]	S. C. Mandal, L. Maganti, M. Mondal, J. Chakrabarti, Biopolymers, 2020, 111, e23396.
[27]	T. Roos, B. Yazdan Panah, M. Kunze,US Patent 20220389470A1, 2022.
[28]	X. Piao, Y. Tang, X. Li, W. Zhang, W. Yang, X. Xu, W. Wang, J. Jiang, J. Xu, K. Hu, M. Xu, M. Liu, M. Sun, L. Jin, Mol. Ther. Nucleic Acids, 2024, 35, 102223.
[29]	J. Martínez, V. Lampaya, A. Larraga, H. Magallón, D. Casabona, Front. Mol. Biosci., 2023, 10, 1248511.
[30]	K. Božič, A. Sedlar, Š. Kralj, U. Černigoj, A. Štrancar, R. Sekirnik, Biotechnol. Bioeng., 2024, 121, 1739-1749.
[31]	W. de Mey, P. De Schrijver, D. Autaers, L. Pfitzer, B. Fant, H. Locy, A. Esprit, L. Lybaert, C. Bogaert, M. Verdonck, K. Thielemans, K. Breckpot, L. Franceschini, Mol. Ther. Nucleic Acids, 2022, 29, 943-954.
[32]	X. Mu, S. Hur, Acc. Chem. Res., 2021, 54, 4012-4023.
[33]	B. Alberts, How Cells Read the Genome: From DNA to Protein, 6 th ed.ed., Taylor and Francis Group, New York, 2015, 299-368.
[34]	W. He, X. Zhang, Y. Zou, J. Li, C. Wang, Y. He, Q. Jin, J. Ye, Molecules, 2024, 29, 2461.
[35]	R. Lenk, W. Kleindienst, G. T. Szabó, M. Baiersdörfer, G. Boros, J. M. Keller, A. J. Mahiny, I. Vlatkovic, Front. Mol. Biosci., 2024, 11, 1426129.
[36]	V. S. Anand, S. S. Patel, J. Biol. Chem., 2006, 281, 35677-35685.
[37]	T. A. Steitz, Curr. Opin. Struct. Biol., 2009, 19, 683-690.
[38]	J. F. Sydow, P. Cramer, Curr. Opin. Struct. Biol., 2009, 19, 732-739.
[39]	M.-Z. Yu, N.-N. Wang, J.-Q. Zhu, Y.-X. Lin, WIREs Nanomed. Nanobiotechnol., 2023, 15, e1894.
[40]	I. Karimi-Sani, Z. Molavi, S. Naderi, S.-H. Mirmajidi, I. Zare, Y. Naeimzadeh, A. Mansouri, A. Tajbakhsh, A. Savardashtaki, A. Sahebkar, J. Nanobiotechnol., 2024, 22, 601.
[41]	S. Linares-Fernández, C. Lacroix, J.-Y. Exposito, B. Verrier, Trends Mol. Med., 2020, 26, 311-323.
[42]	F. J. Triana-Alonso, M. Dabrowski, J. Wadzack, K. H. Nierhaus, J. Biol. Chem., 1995, 270, 6298-6307.
[43]	G. A. Nacheva, A. Berzal-Herranz, Eur. J. Biochem., 2003, 270, 1458-1465.
[44]	X. Mu, E. Greenwald, S. Ahmad, S. Hur, Nucleic Acids Res., 2018, 46, 5239-5249.
[45]	Z. Wu, W. Sun, H. Qi, Vaccines, 2024, 12, 873.
[46]	A. Goyon, S. Tang, S. Fekete, D. Nguyen, K. Hofmann, S. Wang, W. Shatz-Binder, K. I. Fernandez, E. S. Hecht, M. Lauber, K. Zhang, Anal. Chem., 2023, 95, 15017-15024.
[47]	J. Camperi, S. Lippold, L. Ayalew, B. Roper, S. Shao, E. Freund, A. Nissenbaum, C. Galan, Q. Cao, F. Yang, C. Yu, A. Guilbaud, Anal. Chem., 2024, 96, 3886-3897.
[48]	A. Nair, Z. Kis, Front. Mol. Biosci., 2024, 11, 1504876.
[49]	S. Borkotoky, A. Murali, Int. J. Biol. Macromol., 2018, 118, 49-56.
[50]	M. Z. Wu, H. Asahara, G. Tzertzinis, B. Roy, RNA, 2020, 26, 345-360.
[51]	J. Ong, V. Potapov, K.-C. Hung, H. Asahara, S. Chong, G. Tzertzinis,US Patent 12091685B2, 2022.
[52]	W. He, X. Zhang, Y. Zou, J. Li, L. Chang, Y.-C. He, Q. Jin, J. Ye, Front. Bioeng. Biotechnol., 2024, 12, 1356354.
[53]	B. Zhu, S. Tabor, C. C. Richardson, Nucleic Acids Res., 2014, 42, e33.
[54]	H. Xia, B. Yu, Y. Jiang, R. Cheng, X. Lu, H. Wu, B. Zhu, RNA Biol., 2022, 19, 1130-1142.
[55]	M. Miller, O. Alvizo, S. Baskerville, A. Chintala, C. Chng, J. Dassie, J. Dorigatti, G. Huisman, S. Jenne, S. Kadam, N. Leatherbury, S. Lutz, M. Mayo, A. Mukherjee, A. Sero, S. Sundseth, J. Penfield, J. Riggins, X. Zhang, Faraday Discuss., 2024, 252, 431-449.
[56]	J. Guillerez, P. J. Lopez, F. Proux, H. Launay, M. Dreyfus, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 5958-5963.
[57]	H. Wu, T. Wei, B. Yu, R. Cheng, F. Huang, X. Lu, Y. Yan, X. Wang, C. Liu, B. Zhu, RNA Biol., 2021, 18, 451-466.
[58]	A. Dousis, K. Ravichandran, E. M. Hobert, M. J. Moore, A. E. Rabideau, Nat. Biotechnol., 2023, 41, 560-568.
[59]	B. Yu, Y. Chen, Y. Yan, X. Lu, B. Zhu, Nucleic Acids Res., 2024, 52, 8443-8453.
[60]	E. Cavac, L. E. Ramírez-Tapia, C. T. Martin, J. Biol. Chem., 2021, 297, 100999.
[61]	K. MalagodaPathiranage, R. Banerjee, C. T. Martin, Nucleic Acids Res., 2024, 52, 10607-10618.
[62]	D. Plaskon, C. Evensen, K. Henderson, B. Palatnik, T. Ishikuri, H.-C. Wang, S. Doughty, M. Thomas RecordJr, J. Mol. Biol., 2022, 434, 167621.
[63]	J. A. Kern, R. H. Davis, Biotechnol. Prog., 1997, 13, 747-756.
[64]	J. K. Heinonen, R. J. Lahti, Anal. Biochem., 1981, 113, 313-317.
[65]	A. Hachimori, Y. Shiroya, A. Hirato, T. Miyahara, T. Samejima, J. Biochem., 1979, 86, 121-130.
[66]	F. Kanwal, T. Chen, Y. Zhang, A. Simair, R. Cai, N. U. S. Sadaf Zaidi, X. Guo, X. Wei, G. Siegel, C. Lu, Cell. Physiol. Biochem., 2018, 48, 1915-1927.
[67]	N. Pardi, H. Muramatsu, D. Weissman, K. Karikó, Methods Mol. Biol., 2013, 969, 29-42.
[68]	C. J. T. Lewis, T. Pan, A. Kalsotra, Nat. Rev. Mol. Cell Biol., 2017, 18, 202-210.
[69]	S. Vaidyanathan, K. T. Azizian, A. K. M. A. Haque, J. M. Henderson, A. Hendel, S. Shore, J. S. Antony, R. I. Hogrefe, M. S. D. Kormann, M. H. Porteus, A. P. McCaffrey, Mol. Ther. Nucleic Acids, 2018, 12, 530-542.
[70]	H. Moradian, T. Roch, L. Anthofer, A. Lendlein, M. Gossen, Mol. Ther. Nucleic Acids, 2022, 27, 854-869.
[71]	T.-H. Chen, V. Potapov, N. Dai, J. L. Ong, B. Roy, Sci. Rep., 2022, 12, 13017.
[72]	K. D. Nance, J. L. Meier, ACS Cent. Sci., 2021, 7, 748-756.
[73]	K. Karikó, H. Muramatsu, J. Ludwig, D. Weissman, Nucleic Acids Res., 2011, 39, e142.
[74]	T. Ziegenhals, R. Frieling, P. Wolf, K. Göbel, S. Koch, M. Lohmann, M. Baiersdörfer, S. Fesser, U. Sahin, A. N. Kuhn, Front. Mol. Biosci., 2023, 10, 1291045.
[75]	Y. Hadas, N. Sultana, E. Youssef, M. T. K. Sharkar, K. Kaur, E. Chepurko, L. Zangi, Mol. Ther. Methods Clin. Dev., 2019, 14, 300-305.
[76]	K. MalagodaPathiranage, E. Cavac, T.-H. Chen, B. Roy, C. T. Martin, Nucleic Acids Res., 2023, 51, e36.
[77]	Y. Sari, S. Sousa Rosa, J. Jeffries, M. P. C. Marques, Sci. Rep., 2024, 14, 9655.
[78]	J. A. Kern, R. H. Davis, Biotechnol. Prog., 1999, 15, 174-184.
[79]	D. Pregeljc, J. Skok, T. Vodopivec, N. Mencin, A. Krušič, J. Ličen, K. Š. Nemec, A. Štrancar, R. Sekirnik, Biotechnol. Bioeng., 2023, 120, 737-747.
[80]	J. Boman, T. Marušič, T. V. Seravalli, J. Skok, F. Pettersson, K. Š. Nemec, H. Widmark, R. Sekirnik, Biotechnol. Bioeng., 2024, 121, 3415-3427.
[81]	X. Piao, V. Yadav, E. Wang, W. Chang, L. Tau, B. E. Lindenmuth, S. X. Wang, Mol. Ther. Nucleic Acids, 2022, 29, 618-624.
[82]	C. Francis, P. Frida J, B. Thanh-Huong, M. Alicja, K. Artem, P. Jérémie, B. Sven Even, RNA Biol., 2024, 21, 365-370.
[83]	Z. J. Kartje, H. I. Janis, S. Mukhopadhyay, K. T. Gagnon, J. Biol. Chem., 2021, 296, 100175.
[84]	J.-C. Boulain, J. Dassa, L. Mesta, A. Savatier, N. Costa, B. H. Muller, G. L’Hostis, E. A. Stura, A. Troesch, F. Ducancel, Protein Eng. Des. Sel., 2013, 26, 725-734.
[85]	S. Borkotoky, C. Kumar Meena, G. M. Bhalerao, A. Murali, Sci. Rep., 2017, 7, 6290.
[86]	V. V. Gurevich, I. D. Pokrovskaya, T. A. Obukhova, S. A. Zozulya, Anal. Biochem., 1991, 195, 207-213.
[87]	M. Maslak, C. T. Martin, Biochemistry, 1994, 33, 6918-6924.
[88]	X. Ge, D. Luo, J. Xu, PLoS One, 2011, 6, e28707.
[89]	M. Frugier, C. Florentz, M. W. Hosseini, J.-M. Lehn, R. Giegé, Nucleic Acids Res., 1994, 22, 2784-2790.
[90]	M. M. Konarska, P. A. Sharp, Cell, 1989, 57, 423-431.
[91]	Z. Chen, Y. Zhang, Biochem. Biophys. Res. Commun., 2005, 333, 664-670.
[92]	M. Das, D. Dasgupta, FEBS Lett., 1998, 427, 337-340.
[93]	Invitrogen Life Technologies, T7 RNA Polymerase, Technical Bulletin 8033-1, Carlsbad, USA, 2002.
[94]	S. E. McMinn, D. V. Miller, D. Yur, K. Stone, Y. Xu, A. Vikram, S. Murali, J. Raffaele, D. Holland, S.-C. Wang, J. P. Smith, Biochemistry, 2024, 63, 2793-2802.
[95]	N. M. Stover, K. Ganko, R. D. Braatz, Biotechnol. Bioeng., 2024, 121, 2636-2647.
[96]	P. G. Popova, M. A. Lagace, G. Tang, A. K. Blakney, Eur. J. Pharm. Biopharm., 2024, 198, 114247.
[97]	Y. Wang, L. Li, A. T. Sutton, Q. Tu, K. Zhao, E. Wen, J. Osborn, A. Singh, M. J. Gunsch, R. R. Rustandi, D. Foley, Y. He, Anal. Bioanal. Chem., 2024, 416, 2941-2949.
[98]	S. S. Rosa, D. Nunes, L. Antunes, D. M. F. Prazeres, M. P. C. Marques, A. M. Azevedo, Biotechnol. Bioeng., 2022, 119, 3127-3139.
[99]	S. Arnold, M. Siemann, K. Scharnweber, M. Werner, S. Baumann, M. Reuss, Biotechnol. Bioeng., 2001, 72, 548-561.
[100]	P. Thomen, P. J. Lopez, U. Bockelmann, J. Guillerez, M. Dreyfus, F. Heslot, Biophys. J., 2008, 95, 2423-2433.
[101]	S. Akama, M. Yamamura, T. Kigawa, Biophys. J., 2012, 102, 221-230.
[102]	A. Ramanathan, G. B. Robb, S.-H. Chan, Nucleic Acids Res., 2016, 44, 7511-7526.
[103]	F. Muttach, N. Muthmann, A. Rentmeister, Beilstein J. Org. Chem., 2017, 13, 2819-2832.
[104]	O. J. P. Kyrieleis, J. Chang, M. de la Peña, S. Shuman, S. Cusack, Structure, 2014, 22, 452-465.
[105]	R. Kasprzyk, J. Jemielity, ChemBioChem, 2021, 22, 3236-3253.
[106]	P. X. Guo, B. Moss, Proc. Natl. Acad. Sci. USA, 1990, 87, 4023-4027.
[107]	S. H. Chan, C. N. Molé, D. Nye, L. Mitchell, N. Dai, J. Buss, D. W. Kneller, J. M. Whipple, G. B. Robb, RNA, 2023, 29, 1803-1817.
[108]	S. Shuman, J. Hurwitz, Proc. Natl. Acad. Sci. USA, 1981, 78, 187-191.
[109]	Y. Luo, X. Mao, L. Deng, P. Cong, S. Shuman, J. Virol., 1995, 69, 3852-3856.
[110]	M. De la Peña, O. J. P. Kyrieleis, S. Cusack, EMBO J., 2007, 26, 4913-4925.
[111]	L. Yu, S. Shuman, J. Virol., 1996, 70, 6162-6168.
[112]	P. J. Sikorski, M. Warminski, D. Kubacka, T. Ratajczak, D. Nowis, J. Kowalska, J. Jemielity, Nucleic Acids Res., 2020, 48, 1607-1626.
[113]	H. Ohno, S. Akamine, M. Mochizuki, K. Hayashi, S. Akichika, T. Suzuki, H. Saito, Nucleic Acids Res., 2023, 51, e34.
[114]	E. Decroly, B. Canard, Curr. Opin. Virol., 2017, 24, 87-96.
[115]	M. Byszewska, M. Śmietański, E. Purta, J. M. Bujnicki, RNA Biol., 2014, 11, 1597-1607.
[116]	S. W. Lockless, H.-T. Cheng, A. E. Hodel, F. A. Quiocho, P. D. Gershon, Biochemistry, 1998, 37, 8564-8574.
[117]	A.-L. Fuchs, A. Neu, R. Sprangers, RNA, 2016, 22, 1454-1466.
[118]	A. Y. Rudenko, S. S. Mariasina, P. V. Sergiev, V. I. Polshakov, Mol. Biol., 2022, 56, 229-250.
[119]	C. Schuberth-Wagner, J. Ludwig, A. K. Bruder, A.-M. Herzner, T. Zillinger, M. Goldeck, T. Schmidt, J. L. Schmid-Burgk, R. Kerber, S. Wolter, J.-P. Stümpel, A. Roth, E. Bartok, C. Drosten, C. Coch, V. Hornung, W. Barchet, B. M. Kümmerer, G. Hartmann, M. Schlee, Immunity, 2015, 43, 41-51.
[120]	S. C. Devarkar, C. Wang, M. T. Miller, A. Ramanathan, F. Jiang, A. G. Khan, S. S. Patel, J. Marcotrigiano, Proc. Natl. Acad. Sci. USA, 2016, 113, 596-601.
[121]	J.-B. Marq, S. Hausmann, N. Veillard, D. Kolakofsky, D. Garcin, J. Biol. Chem., 2011, 286, 6108-6116.
[122]	S. Linares-Fernández, J. Moreno, E. Lambert, P. Mercier-Gouy, L. Vachez, B. Verrier, J.-Y. Exposito, Mol. Ther. Nucleic Acids, 2021, 26, 945-956.
[123]	T. Roos, B. Y. Panah, M. Conzelmann, A. Thess, D. Buob, M. Kunze, V. Wagner,WO Patent 2016193226A1, 2016.
[124]	G. B. Robb, S.-H. Chan, B. Roy,US Patent 20210054016A1, 2021.
[125]	H. Lönnberg, Chem. Rec., 2022, 22, e202200141.
[126]	R. T. Raines, Chem. Rev., 1998, 98, 1045-1066.
[127]	U. Arnold, R. Ulbrich-Hofmann, J. Protein Chem., 2000, 19, 345-352.
[128]	E. E. Hameş, T. Demir, World J. Microbiol. Biotechnol., 2015, 31, 1853-1862.
[129]	E. A. Ponomareva, M. V. Volokitina, D. O. Vinokhodov, E. G. Vlakh, T. B. Tennikova, Anal. Bioanal. Chem., 2013, 405, 2195-2206.
[130]	W.-J. Chen, T.-H. Liao, Protein Pept. Lett., 2006, 13, 447-453.
[131]	M. Guéroult, D. Picot, J. Abi-Ghanem, B. Hartmann, M. Baaden, PLoS Comput. Biol., 2010, 6, e1001000.
[132]	P. Pourali, V. Dzmitruk, M. Patek, E. Neuhoferova, M. Svoboda, V. Benson, Sci. Rep., 2023, 13, 4916.
[133]	L.-Y. Chu, T.-J. Hsieh, B. Golzarroshan, Y.-P. Chen, S. Agrawal, H. S. Yuan, Nucleic Acids Res., 2017, 45, 12015-12024.
[134]	D. H. Bechhofer, M. P. Deutscher, Crit. Rev. Biochem. Mol. Biol., 2019, 54, 242-300.
[135]	A. W. Nicholson, WIREs RNA, 2014, 5, 31-48.
[136]	M. Baiersdörfer, G. Boros, H. Muramatsu, A. Mahiny, I. Vlatkovic, U. Sahin, K. Karikó, Mol. Ther. Nucleic Acids, 2019, 15, 26-35.
[137]	D. Niu, Y. Wu, J. Lian, Signal Transduct. Target. Ther., 2023, 8, 341.
[138]	R. A. Wesselhoeft, P. S. Kowalski, F. C. Parker-Hale, Y. Huang, N. Bisaria, D. G. Anderson, Mol. Cell, 2019, 74, 508-520.e4.
[139]	X. Xiao, D. Yin, C. Hua,CN Patent 116286796A, 2023.
[140]	M. Shanmugasundaram, A. Senthilvelan, A. R. Kore, Chem. Rec., 2022, 22, e202200005.
[141]	N. Pardi, M. J. Hogan, D. Weissman, Curr. Opin. Immunol., 2020, 65, 14-20.
[142]	H. Kwon, M. Kim, Y. Seo, Y. S. Moon, H. J. Lee, K. Lee, H. Lee, Biomaterials, 2018, 156, 172-193.
[143]	R. Grzela, K. Piecyk, A. Stankiewicz-Drogon, P. Pietrow, M. Lukaszewicz, K. Kurpiejewski, E. Darzynkiewicz, M. Jankowska-Anyszka, RNA, 2023, 29, 200-216.
[144]	E. Grudzien-Nogalska, J. Stepinski, J. Jemielity, J. Zuberek, R. Stolarski, R. E. Rhoads, E. Darzynkiewicz, Synthesis of Anti-reverse Cap Analogs (ARCAs) and Their Applications in mRNA Translation and Stability, 1st ed.ed., Elsevier Science & Technology, Cambridge, 2007, 203-227.
[145]	A. O. Nwokeoji, T. Chou, E. A. Nwokeoji, ACS Synth. Biol., 2023, 12, 329-339.
[146]	H. Helgers, A. Hengelbrock, A. Schmidt, J. Strube, Processes, 2021, 9, 1967.
[147]	L. Guo, Z. Liu, S. Song, W. Yao, M. Yang, G. Chen, Biochem. Eng. J., 2024, 210, 109412.
[148]	A. C. Fisher, M.-H. Kamga, C. Agarabi, K. Brorson, S. L. Lee, S. Yoon, Trends Biotechnol., 2019, 37, 253-267.
[149]	A. Ouranidis, C. Davidopoulou, R.-K. Tashi, K. Kachrimanis, Pharmaceutics, 2021, 13, 1371.
[150]	F. L. Vetter, S. Zobel-Roos, J. P. B. Mota, B. Nilsson, A. Schmidt, J. Strube, Processes, 2022, 10, 1868.
[151]	Y. Liu, E. Holmstrom, J. Zhang, P. Yu, J. Wang, M. A. Dyba, D. Chen, J. Ying, S. Lockett, D. J. Nesbitt, A. R. Ferré-D’Amaré, S. Rui, J. R. Stagno, Y.-X. Wang, Nature, 2015, 522, 368-372.
[152]	Y. Thillier, E. Decroly, F. Morvan, B. Canard, J.-J. Vasseur, F. Debart, RNA, 2012, 18, 856-868.
[153]	S. Che, X. Feng, Z. Li, Z. Su, G. Ma, Z. Li, A. Yu, M. Liu, S. Zhang, Biotechnol. Bioeng., 2024, 121, 206-218.
[154]	B. J. Engel, B. J. Grindel, J. P. Gray, S. W. Millward, Bioorg. Med. Chem. Lett., 2020, 30, 126934.
[155]	V. D’Atri, M. Imiołek, C. Quinn, A. Finny, M. Lauber, S. Fekete, D. Guillarme, J. Chromatogr. A, 2024, 1722, 464862.
[156]	N. M. Stover, S. Ahmadi, J. Rosenfeld, F. Destro, A. S. Myerson, R. D. Braatz, ChemRxiv, 2025, 1-29.
[157]	C. He, C. Zhang, T. Bian, K. Jiao, W. Su, K.-J. Wu, A. Su, Processes, 2023, 11, 330.
[158]	N. A. Yewdall, A. A. M. André, T. Lu, E. Spruijt, Curr. Opin. Colloid Interface Sci., 2021, 52, 101416.
[159]	E. Kobatake, A. Ebisawa, O. Asaka, Y. Yanagida, Y. Ikariyama, M. Aizawa, Appl. Biochem. Biotechnol., 1999, 76, 217-228.
[160]	Y. Wang, C. Wang, Y. Zheng, H. Hu, Y. Chen, Y. Lu, J. Am. Chem. Soc., 2025, 147, 21926-21939.
[161]	A. Schmidt, H. Helgers, F. L. Vetter, S. Zobel-Roos, A. Hengelbrock, J. Strube, Processes, 2022, 10, 1783.
[162]	V. Sharma, J.-U. Joo, A. Mottafegh, D.-P. Kim, Chem. Eng. J., 2025, 517, 164284.

Engineering of enzymatic modules for mRNA manufacturing: Advances in catalytic regulation and process integration

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

share this article

Figures/Tables 13

References

Related Articles 0

Recommended Articles

Metrics