An engineered T7 RNA polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts in mRNA synthesis

Miller, Mathew; Alvizo, Oscar; Baskerville, Scott; Chintala, Avinash; Chng, Chinping; Dassie, Justin; Dorigatti, Jonathan; Huisman, Gjalt; Jenne, Stephan; Kadam, Supriya; Leatherbury, Neil; Lutz, Stefan; Mayo, Melissa; Mukherjee, Arpan; Sero, Antoinette; Sundseth, Stuart; Penfield, Jonathan; Riggins, James; Zhang, Xiyun

doi:10.1039/d4fd00023d

Cited by 4 publications

(3 citation statements)

References 38 publications

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“…The malachite green assay kit (MAK307, Sigma-Aldrich) was purchased and used according to the manufacturer's protocol. A series of phosphate standard solutions with final concentrations of 40,32,24,16,12,8,4, and 0 μM, containing malachite green, were prepared, and their absorbance was measured at 644 nm. The protein crude product, prepared using the previously described recipe, was diluted to fall within the appropriate measurement range, and its absorbance was measured.…”

Section: Generation Of Egfp and 4-mu Calibration Curvesmentioning

confidence: 99%

“…as engineered lipases and proteases for detergents, novel catalysts for bioremediation and green chemistry, and degradative enzymes for cellulosic biofuels [1][2][3][4][5][6][7][8][9][10] ) and medicine (e.g. as targets for drugs used to treat cancer [11][12][13][14][15][16] and infectious disease [17][18][19][20][21] , as catalysts for efficient synthesis of pharmaceuticals [22][23][24][25] , and as therapeutics in metabolic flux disorders [26][27][28][29] ). Despite impressive and rapid progress in the ability to design protein sequences that reliably adopt desired low-energy structures, designing enzymes with specified activities, specificities, and catalytic efficiencies remains an unsolved challenge.…”

Section: Introductionmentioning

confidence: 99%

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Hydrophilic/ Omniphobic droplet arrays for high-throughput and quantitative enzymology

Lee,

Sunden,

Miller

et al. 2024

Preprint

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Engineered enzymes with enhanced or novel functions are specific catalysts with wide-ranging applications in industry and medicine. Here, we introduce Droplet Array Microfluidic Enzyme Kinetics (DA-MEK), a high-throughput enzyme screening platform that combines water-in-air droplet microarrays formed on patterned superhydrophilic/omniphobic surfaces with cell-free protein synthesis to enable cost-effective expression and quantitative kinetic characterization of enzyme variants. By printing DNA templates encoding enzyme variants onto hydrophilic spots, stamping slides to add cell-free expression mix, and imaging the resulting arrays, we demonstrate reproducible expression of hundreds of enzyme variants per slide. Subsequent stamping of fluorogenic substrates and time-lapse imaging allows determination of Michaelis-Menten parameters for each variant, with measured catalytic efficiencies spanning 5 orders of magnitude and agreeing well with values obtained via traditional microtiter plate assays. DA-MEK consumes orders of magnitude less reagents than plate-based assays while providing accurate and detailed kinetic information for both beneficial and deleterious mutations. In future work, we anticipate DA-MEK will provide a powerful and versatile platform to accelerate enzyme engineering and enable screening of large variant libraries under diverse conditions.

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Section: Generation Of Egfp and 4-mu Calibration Curvesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Hydrophilic/ Omniphobic droplet arrays for high-throughput and quantitative enzymology

Lee,

Sunden,

Miller

et al. 2024

Preprint

Self Cite

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“…Nevertheless, there is still a lack of dened concentration limits that are established by the regulatory agencies for dsRNA. 3 From a reaction optimisation perspective, multiple approaches have been followed to reduce dsRNA such as engineering T7 RNA polymerase, 13,14 or blocking the 3 0 end with complementary oligonucleotides to avoid overextension. 15 New chromatographic modalities can be applied, exploring physicochemical differences between mRNA and dsRNA.…”

Section: Introductionmentioning

confidence: 99%

A (RP)UHPLC/UV analytical method to quantify dsRNA during the mRNA vaccine manufacturing process

Rosa,

Zhang,

Sari

et al. 2024

Anal. Methods

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Directed evolution of an orthogonal transcription engine for programmable gene expression in eukaryotes

Kar,

Gardner,

Javanmardi

et al. 2024

Preprint

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T7 RNA polymerase has enabled orthogonal control of gene expression and recombinant protein production across diverse prokaryotic host chassis organisms for decades. However, the absence of 5' methyl guanosine caps on T7 RNAP derived transcripts has severely limited its utility and widespread adoption in eukaryotic systems. To address this shortcoming, we evolved a fusion enzyme combining T7 RNAP with the single subunit capping enzyme from African swine fever virus using Saccharomyces cerevisiae. We isolated highly active variants of this fusion enzyme, which exhibited roughly two orders of magnitude higher protein expression compared to the wild-type enzyme. We demonstrate the programmable control of gene expression using T7 RNAP-based genetic circuits in yeast and validate enhanced performance of these engineered variants in mammalian cells. This study presents a robust, orthogonal gene regulatory system applicable across diverse eukaryotic hosts, enhancing the versatility and efficiency of synthetic biology applications.

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An engineered T7 RNA polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts in mRNA synthesis

Abstract: Messenger RNA (mRNA) therapies have recently gained tremendous traction with the approval of mRNA vaccines for the prevention of SARS-CoV-2 infection. However, manufacturing challenges have complicated large scale mRNA production,...

Cited by 4 publications

References 38 publications

Hydrophilic/ Omniphobic droplet arrays for high-throughput and quantitative enzymology

Hydrophilic/ Omniphobic droplet arrays for high-throughput and quantitative enzymology

A (RP)UHPLC/UV analytical method to quantify dsRNA during the mRNA vaccine manufacturing process

Directed evolution of an orthogonal transcription engine for programmable gene expression in eukaryotes

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