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Pyrophosphatase enzymes

Manufactured by Aldevron
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About the product

Pyrophosphatase enzymes are essential laboratory tools used to catalyze the hydrolysis of pyrophosphate (PPi) into two phosphate ions. These enzymes play a crucial role in maintaining the equilibrium of phosphate-based reactions, making them a valuable component in various biochemical and molecular biology applications.

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Lab products found in correlation

Rnase inhibitor, by Aldevron (5 mentions) S adenosyl methionine, by Aldevron (4 mentions) T7 polymerase, by Aldevron (3 mentions) Capto, by Cytiva (3 mentions) Capto core 700 instrument, by GE Healthcare (2 mentions) Bspqi restriction enzyme, by New England Biolabs (2 mentions) Capto core 700 resin, by GE Healthcare (2 mentions) Dnase 1, by Aldevron (2 mentions) Ribogreen assay, by Thermo Fisher Scientific (2 mentions) S adenosylmethionine, by New England Biolabs (1 mentions) Bspqi enzymes, by New England Biolabs (1 mentions) Vaccinia capping enzyme, by Aldevron (1 mentions) Rntp mix, by New England Biolabs (1 mentions) Guanylyl transferase, by Aldevron (1 mentions) Mwco modified polyethersulfone mpes membrane, by Repligen (1 mentions)
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5 protocols using «pyrophosphatase enzymes»

1

In Vitro Transcription of saRNA

2022
Vaccine saRNA was generated by T7 polymerase-mediated in vitro transcription (IVT) using NotIlinearized DNA plasmids as templates. An in-house optimized IVT protocol was used with commercially available rNTP mix (NEB) and commercially available T7 polymerase, RNase inhibitor, and pyrophosphatase enzymes (Aldevron, Fargo, ND). DNA plasmids were digested away (DNase I, Aldevron), and Cap 0 structures were added to the transcripts by treatment with guanylyltransferase (Aldevron), GTP, and S-adenosylmethionine (NEB). RNA was chromatographically purified using Capto Core 700 resin (GE Healthcare, Chicago, IL) followed by diafiltration and concentration through tangential flow filtration using a 750 kDa molecular weight cut-off (MWCO) modified polyethersulfone (mPES) membrane (Repligen, Waltham, MA). Terminal filtration of the saRNA material was done using a 0.22 µm PES filter, and the saRNA materials were stored at -80°C until use/complexation. Agarose gel electrophoresis was used to characterize saRNA size and integrity, and RNA concentration was quantified by UV absorbance (NanoDrop 1000) and RiboGreen assay (Thermo Fisher Scientific, Waltham, MA).
Voigt E.A., Gerhardt A., Hanson D., Battisti P., Reed S., Singh J., Mohamath R., Jennewein M.F., Bakken J., Beaver S., Press C., Soon‐Shiong P., Paddon C.J., Fox C.B., & Casper C. (2022). A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability.
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2

Generation of Stabilized mRNA Stocks

2022
Generation of saRNA stocks was achieved by T7 promoter-mediated in vitro transcription using NotI-linearized DNA template. In vitro transcription was performed using an in-house-optimized protocol using T7 polymerase, RNase inhibitor, and pyrophosphatase enzymes procured from Aldevron. DNA plasmid was digested away (DNase I, Aldevron) and cap0 structures were added to the transcripts by vaccinia capping enzyme, GTP, and S-adenosyl-methionine (Aldevron). RNA was then purified from the transcription and capping reaction components by chromatography using a CaptoCore 700 resin (GE Healthcare) followed by diafiltration and concentration using tangential flow filtration. The saRNA material was terminally filtered with a 0.22 μm polyethersulfone filter and stored at −80°C until use. All saRNA was characterized by agarose gel electrophoresis and quantified both by UV absorbance (NanoDrop 1000) and Ribogreen assay (Thermo Fisher). OVA-expressing mRNA was obtained from a commercial vendor (TriLink CleanCap OVA mRNA, L-7610).
Gerhardt A., Voigt E., Archer M., Reed S., Larson E., Van Hoeven N., Kramer R., Fox C, & Casper C. (2022). A flexible, thermostable nanostructured lipid carrier platform for RNA vaccine delivery. Molecular Therapy. Methods & Clinical Development, 25, 205-214.
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3

Scalable RNA Production for Vaccine Research

2020
cDNAs encoding the 20 lead mAb candidates were amplified from the mammalian expression pTwist vector using universal primer sets and cloned into plasmids encoding Venezuelan equine encephalitis virus replicon (strain TC-83) under the control of a T7 promoter (pT7-VEE-Rep)32 (link). To generate linear templates for RNA transcription for RNA in vitro transcription and capping at midi scale (500 μg), plasmid DNA was cleaved using NotI or BspQI restriction enzymes (New England Biolabs), respectively, and purified using phenol-chloroform extraction and sodium acetate precipitation. RNA was transcribed using T7 Polymerase, RNAse inhibitor, Pyrophosphatase enzymes (Aldevron) and reaction buffer. RNA transcripts were capped with vaccinia virus capping enzyme using GTP and S-adenosyl-methionine (Aldevron) as substrates to create cap-0 structures. RNA was purified using lithium chloride precipitation. Large-scale capability demonstration RNA production was performed using a 200 mg scaled-up process and purified via column on a Capto Core 700 instrument (GE Healthcare Life Sciences) and tangential flow filtration.
Gilchuk P., Bombardi R.G., Erasmus J.H., Tan Q., Nargi R., Soto C., Abbink P., Suscovich T.J., Durnell L.A., Khandhar A., Archer J., Liang J., Fouch M.E., Davidson E., Doranz B.J., Jones T., Larson E., Ertel S., Granger B., Fuerte-Stone J., Roy V., Broge T., Linnekin T.C., Linde C.H., Gorman M.J., Nkolola J., Alter G., Reed S.G., Barouch D.H., Diamond M.S., Crowe JE J.r., Van Hoeven N., Thackray L, & Carnahan R. (2020). Integrated pipeline for the accelerated discovery of antiviral antibody therapeutics. Nature biomedical engineering, 4(11), 1030-1043.
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4

RNA Transcription Template Generation

2020
To generate linear templates for RNA transcription, we cut repRNA or mRNA plasmid DNA by restriction digest using NotI or BspQI enzymes (New England Biolabs), respectfully, and purified using phenol-chloroform extraction and sodium acetate precipitation. RNA was transcribed using T7 polymerase, RNase inhibitor, pyrophosphatase enzymes (Aldevron), and reaction buffer. RNA transcripts were capped with vaccinia virus capping enzyme using GTP and S-adenosyl-methionine (New England Biolabs) as substrates to create a cap-0 structure. RNA was purified using LiCl precipitation. To generate pseudouridine-modified RNAs, was replaced 100% of uridine with pseudouridine in the reaction mixture and transcribed and capped as described above.
Erasmus J.H., Archer J., Fuerte-Stone J., Khandhar A.P., Voigt E., Granger B., Bombardi R.G., Govero J., Tan Q., Durnell L.A., Coler R.N., Diamond M.S., Crowe JE J.r., Reed S.G., Thackray L.B., Carnahan R.H, & Van Hoeven N. (2020). Intramuscular Delivery of Replicon RNA Encoding ZIKV-117 Human Monoclonal Antibody Protects against Zika Virus Infection. Molecular Therapy. Methods & Clinical Development, 18, 402-414.
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5

Scalable RNA Production for Vaccine Research

2020
cDNAs encoding the 20 lead mAb candidates were amplified from the mammalian expression pTwist vector using universal primer sets and cloned into plasmids encoding Venezuelan equine encephalitis virus replicon (strain TC-83) under the control of a T7 promoter (pT7-VEE-Rep)32 (link). To generate linear templates for RNA transcription for RNA in vitro transcription and capping at midi scale (500 μg), plasmid DNA was cleaved using NotI or BspQI restriction enzymes (New England Biolabs), respectively, and purified using phenol-chloroform extraction and sodium acetate precipitation. RNA was transcribed using T7 Polymerase, RNAse inhibitor, Pyrophosphatase enzymes (Aldevron) and reaction buffer. RNA transcripts were capped with vaccinia virus capping enzyme using GTP and S-adenosyl-methionine (Aldevron) as substrates to create cap-0 structures. RNA was purified using lithium chloride precipitation. Large-scale capability demonstration RNA production was performed using a 200 mg scaled-up process and purified via column on a Capto Core 700 instrument (GE Healthcare Life Sciences) and tangential flow filtration.
Gilchuk P., Bombardi R.G., Erasmus J.H., Tan Q., Nargi R., Soto C., Abbink P., Suscovich T.J., Durnell L.A., Khandhar A., Archer J., Liang J., Fouch M.E., Davidson E., Doranz B.J., Jones T., Larson E., Ertel S., Granger B., Fuerte-Stone J., Roy V., Broge T., Linnekin T.C., Linde C.H., Gorman M.J., Nkolola J., Alter G., Reed S.G., Barouch D.H., Diamond M.S., Crowe JE J.r., Van Hoeven N., Thackray L, & Carnahan R. (2020). Integrated pipeline for the accelerated discovery of antiviral antibody therapeutics. Nature biomedical engineering, 4(11), 1030-1043.
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