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Scriptcap 2 o methyltransferase kit

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The ScriptCap™ 2′-O-Methyltransferase Kit is a laboratory tool designed for the 2'-O-methylation of the 5' cap structure of in vitro transcribed RNA. It contains the necessary components to perform this enzymatic capping reaction.

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Lab products found in correlation

Scriptcap m7g capping system, by CellScript (7 mentions) Nanodrop one, by Thermo Fisher Scientific (4 mentions) Megascript t7 transcription kit, by Thermo Fisher Scientific (3 mentions) T7 scribe standard rna ivt kit, by CellScript (2 mentions) Megascript, by Thermo Fisher Scientific (2 mentions) Plasmid plus maxiprep kit, by Qiagen (2 mentions) Carbenicillin, by Merck Group (2 mentions) Neon electroporation kit, by Thermo Fisher Scientific (2 mentions) T7 rna polymerase, by Thermo Fisher Scientific (2 mentions) Rneasy mini kit, by Qiagen (1 mentions) Mouse erythropoietin quantikine elisa kit, by R&D Systems (1 mentions) M1ψ 5 triphosphate, by TriLink (1 mentions) 1 2 dioleoyl sn glycero 3 phosphoethanolamine dope, by Avanti Polar Lipids (1 mentions) C14 peg2000, by Avanti Polar Lipids (1 mentions) Xenolight d luciferin potassium salt, by PerkinElmer (1 mentions)

12 protocols using scriptcap 2 o methyltransferase kit

1

In Vitro Transcription of RNA

Cited 1 time
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The DNA fragments PCR-amplified from psiCHECK2−7×AGAGAG motifs or psiCHECK2-CAA repeats (Iwasaki et al., 2016 (link)) were used as a template for in vitro transcription with a T7-Scribe Standard RNA IVT Kit (CELLSCRIPT). RNA was capped and poly(A) tailed with a ScriptCap m7G Capping System, a ScriptCap 2′-O-Methyltransferase Kit, and an A-Plus Poly(A) Polymerase Tailing Kit (CELLSCRIPT).
Chen M., Kumakura N., Saito H., Muller R., Nishimoto M., Mito M., Gan P., Ingolia N.T., Shirasu K., Ito T., Shichino Y, & Iwasaki S. (2023). A parasitic fungus employs mutated eIF4A to survive on rocaglate-synthesizing Aglaia plants. eLife, 12, e81302.
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2

Synthetic VEE Replicon RNA Production

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Example 3

The Alphaviral replicon DNA template for in vitro transcription (IVT) was generated by Gibson assembly using synthetic DNA fragments based on the sequence of the Venezuelan equine encephalitis (VEE) TC-83 strain containing a A3G mutation in the 5′UTR. VEE replicon RNA was produced by run-off IVT of I-SceI-digested replicon plasmid DNA using the MEGAscript® T7 Transcription Kit, followed by purification using the RNeasy® Mini Kit (Qiagen), denaturation of the RNA at 65 degrees C., enzymatic (cap1) capping of the RNA using the ScriptCap™ 2′-O-Methyltransferase Kit (Cellscript) and ScriptCap™ m7G Capping System (Cellscript), and a final purification using the RNeasy® Mini Kit (Qiagen) following the manufacturers' protocols.

US11026894B2. Lipid nanoparticles and use thereof to deliver RNA polynucleotides (2021-06-08). Massachusetts Institute of Technology [US]. Inventors: Darrell J. Irvine [US], Ron Weiss [US], Tasuku Kitada [US], Mariane Melo [US], Yuan Zhang [US].
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3

Modified mRNA Production for HIV-1 Vaccine

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Modified mRNAs were produced by in vitro transcription using T7 RNA polymerase (Megascript, Ambion) on linearized plasmids encoding codon-optimized CH848 10.17DT gp160s, CH848 10.17DT SOSIP trimers, or CH848 10.17DT trimer-ferritin NPs. All the HIV-1 modified mRNA constructs used in this study and their corresponding plasmids were listed in Table S1. One-methylpseudouridine (m1Ψ)-5’-triphosphate (TriLink, Cat# N-1081), instead of UTP was used to produce nucleoside-modified mRNAs. Modified mRNAs contain 101 nucleotide-long polyadenylation tails for optimized expression. Modified CH848 10.17DT SOSIPv4.1 trimer and CH848 10.17DT SOSIPv5.2.8 trimer mRNAs were capped using ScriptCap m7G capping system and ScriptCap 2′-O-methyl-transferase kit (ScriptCap, CellScript) (Pardi et al., 2013 (link)). Capping of all other in vitro transcribed mRNAs was performed co-transcriptionally using the trinucleotide cap1 analog, CleanCap (TriLink, Cat# N-7413). All mRNAs were purified by cellulose purification, as described (Baiersdorfer et al., 2019 (link)). All mRNAs were analyzed by agarose gel electrophoresis and were stored frozen at −20 °C.
Mu Z., Wiehe K., Saunders K.O., Henderson R., Cain D.W., Parks R., Martik D., Mansouri K., Edwards R.J., Newman A., Lu X., Xia S.M., Bonsignori M., Montefiori D., Han Q., Venkatayogi S., Evangelous T., Wang Y., Rountree W., Tam Y., Barbosa C., Alam S.M., Williams W.B., Pardi N., Weissman D, & Haynes B.F. (2021). Ability of nucleoside-modified mRNA to encode HIV-1 envelope trimer nanoparticles. bioRxiv.
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4

Synthesis of Modified mRNA and Lipid Nanoparticles

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Cy5-labeled mRNA, ψ- 5’-triphosphate, m1ψ- 5’-triphosphate, m5C- 5’-triphosphate, s2U- 5’-triphosphate, and m5U- 5’-triphosphate were purchased from Trilink Biotechnologies (San Diego, CA). MEGAscript™ T7 Transcription Kit was purchased from Thermo Fisher Scientific (Waltham, MA). ScriptCap™ 2’-O-Methyltransferase Kit was purchased from CellScript (Madison, WI). Cholesterol and 1,2-epoxyhexadecane (C12) were purchased from Sigma Aldrich (St Louis, MO). The phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and C14-PEG2000 were purchased from Avanti Polar Lipids (Alabaster, AL). DMG-PEG2000 was purchased from NOF America (White Plains, NY). The isodecyl acrylate (Oi10) amine and the 2[4-2(2-aminoethyl)amino)ethylpiperazine-1-YL)ethan-1-amine (200) were purchased from Sartomer (Colombes, France) and Enamine (Princeton, NJ), respectively. XenoLight D-Luciferin Potassium Salt was purchased from PerkinElmer (Waltham, MA). The lipid cKK-E12 was generously donated by the Anderson Lab at the Massachusetts Institute of Technology (Cambridge, MA). The Mouse Erythropoietin Quantikine ELISA Kit was purchased from R&D Systems (Minneapolis, MN).
Melamed J.R., Hajj K.A., Chaudhary N., Strelkova D., Arral M.L., Pardi N., Alameh M.G., Miller J.B., Farbiak L., Siegwart D.J., Weissman D, & Whitehead K.A. (2021). Lipid nanoparticle chemistry determines how nucleoside base modifications alter mRNA delivery. Journal of controlled release : official journal of the Controlled Release Society, 341, 206-214.
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5

Renilla Luciferase Reporter mRNA Preparation

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Reporter mRNA of Renilla luciferase with CAA repeats or 7×AGAGAG motifs was prepared as previously described (Iwasaki et al., 2016 (link)). Briefly, the DNA fragment was PCR-amplified from psiCHECK2-CAA repeats or psiCHECK2-7×AGAGAG motifs (Iwasaki et al., 2016 (link)) and used for in vitro transcription with a T7-Scribe Standard RNA IVT Kit (CELLSCRIPT), after which capping and polyadenylation were performed with a ScriptCap m7G Capping System, a ScriptCap 2′-O-Methyltransferase Kit, and an A-Plus Poly(A) Polymerase Tailing Kit (CELLSCRIPT).
Chen M., Asanuma M., Takahashi M., Shichino Y., Mito M., Fujiwara K., Saito H., Floor S.N., Ingolia N.T., Sodeoka M., Dodo K., Ito T, & Iwasaki S. (2020). Dual targeting of DDX3 and eIF4A by the translation inhibitor rocaglamide A. Cell chemical biology.
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6

VEEV-Based Self-Amplifying RNA Production

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Self-amplifying RNA
derived from the Venezuelan Equine Encephalitis Virus (VEEV) encoding
either firefly luciferase (fLuc) or enhanced green fluorescent protein
(eGFP) was prepared using in vitro transcription.
pDNA was transformed in Escherichia coli and cultured
in 50 mL of LB with 1 mg/mL carbenicillin (Sigma–Aldrich, U.K.)
and isolated using a Plasmid Plus Maxiprep kit (QIAGEN, U.K.). pDNA
concentration and purity was measured on a NanoDrop One (ThermoFisher,
U.K.) and then linearized using MluI for 2 h at 37 °C and heat
inactivated at 80 °C for 20 min. Uncapped in vitro RNA transcripts
were synthesized using 1 μg of linearized DNA template in a
MEGAScript reaction (Promega, U.K.), according to the manufacturer’s
protocol. Transcripts were then purified by overnight LiCl precipitation
at −20 °C, pelleted by centrifugation at 14 000
rpm for 20 min, washed once with 70% EtOH, centrifuged at 14 000
rpm for 5 min, and then resuspended in UltraPure H2O. Purified
transcripts were then capped using the ScriptCap m7G Capping System (CellScript, Madison, WI, USA) and ScriptCap 2′-O-Methyltransferase
Kit (CellScript, Madison, WI, USA) simultaneously, according to the
manufacturer’s protocol. Capped transcripts were then purified
again by LiCl precipitation, resuspended in ultraPure H2O, and stored at −80 °C until use.
Blakney A.K., McKay P.F., Ibarzo Yus B., Hunter J.E., Dex E.A, & Shattock R.J. (2019). The Skin You Are In: Design-of-Experiments Optimization of Lipid Nanoparticle Self-Amplifying RNA Formulations in Human Skin Explants. ACS Nano, 13(5), 5920-5930.
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7

VEE Replicon Plasmid DNA Preparation

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VEE replicon plasmid DNA was prepared based on mutant constructs previously described16 (link). The sequence of the newly discovered mutant “dead” replicon deRep are shown in Supplementary Table 2. mCherry, EGFP or firefly luciferase were cloned after the subgenomic promoter for reporter constructs to generate reporter constructs as previously described16 (link). IL-12-alb and IL-12-alb-lum fusion payload genes with sequences as previously described26 (link) were cloned after the subgenomic promoter to generate therapeutic replicons. Replicon RNAs were in vitro transcribed (IVT) from the templates of linearized VEE DNA constructs using the MEGAscript™ T7 Transcription Kit (ThermoFisher) following the manufacturer’s instructions. The resulting replicon RNAs were capped and methylated using the ScriptCap™ m7G Capping System and ScriptCap™ 2’-O-Methyltransferase Kit (Cellscript) according to the manufacturer’s instructions. RNA purity was assessed by gel electrophoresis. In vitro transfections were carried out using electroporation with 5 μg RNA per 500,000 cells in 100 μl R buffer using a NEON electroporation kit (ThermoFisher) at 1200 V, 20 milliseconds, and 1 pulse.
Li Y., Su Z., Zhao W., Zhang X., Momin N., Zhang C., Wittrup K.D., Dong Y., Irvine D.J, & Weiss R. (2020). Multifunctional oncolytic nanoparticles deliver self-replicating IL-12 RNA to eliminate established tumors and prime systemic immunity. Nature cancer, 1(9), 882-893.
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8

mRNA Vaccine Manufacturing Process

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Antigens encoded by the mRNA vaccines were derived from the ancestral SARS-CoV-2 Wuhan-Hu-1 strain (GenBank MN908947.3). Nucleoside-modified mRNAs expressing SARS-CoV-2 full-length N (mRNA-N) or prefusion-stabilized S protein with two proline mutations (mRNA-S-2P) were synthesized by in vitro transcription using T7 RNA polymerase (MegaScript, Thermo Fisher Scientific) on linearized plasmid templates, as previously reported (26 (link)). Uridine triphosphate was replaced with one-methylpseudouridine (m1Ψ)-5′-triphosphate (TriLink, catalog no. N-1081) for producing nucleoside-modified mRNAs. Polyadenylated tails were added to the end of modified mRNAs for optimized protein expression. In vitro transcribed mRNAs were capped using ScriptCap m7G capping system and ScriptCap 2′-O-methyltransferase kit (ScriptCap, CellScript) (26 (link)), followed by purification using the cellulose purification method as previously described (27 (link)). Purified mRNAs were analyzed by agarose gel electrophoresis and were kept frozen at −20°C. The mRNAs were formulated into LNPs using an ethanolic lipid mixture of ionizable cationic lipid and an aqueous buffer system, as previously reported (28 (link), 57 (link)). Formulated mRNA-LNPs were prepared according to RNA concentrations (1 μg/μl) and were stored at −80°C for animal immunizations.
Hajnik R.L., Plante J.A., Liang Y., Alameh M.G., Tang J., Bonam S.R., Zhong C., Adam A., Scharton D., Rafael G.H., Liu Y., Hazell N.C., Sun J., Soong L., Shi P.Y., Wang T., Walker D.H., Sun J., Weissman D., Weaver S.C., Plante K.S, & Hu H. (2022). Dual spike and nucleocapsid mRNA vaccination confer protection against SARS-CoV-2 Omicron and Delta variants in preclinical models. Science translational medicine, 14(662), eabq1945.
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9

Production and Purification of Modified HIV-1 mRNAs

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Modified mRNAs were produced by in vitro transcription using T7 RNA polymerase (Megascript, Ambion) on linearized plasmids encoding codon-optimized CH848 10.17DT gp160s, CH848 10.17DT SOSIP trimers, or CH848 10.17DT trimer-ferritin NPs. All the HIV-1 modified mRNA constructs used in this study and their corresponding plasmids were listed in Table S2. One-methylpseudouridine (m1Ψ)-5′-triphosphate (TriLink, Cat# N-1081), instead of UTP was used to produce nucleoside-modified mRNAs. Modified mRNAs contain 101 nucleotide-long polyadenylation tails for optimized expression. Modified CH848 10.17DT SOSIPv4.1 trimer and CH848 10.17DT SOSIPv5.2.8 trimer mRNAs were capped using ScriptCap m7G capping system and ScriptCap 2′-O-methyl-transferase kit (ScriptCap, CellScript) (Pardi et al., 2013 (link)). Capping of all other in vitro transcribed mRNAs was performed co-transcriptionally using the trinucleotide cap1 analog, CleanCap (TriLink, Cat# N-7413). All mRNAs were purified by cellulose purification, as described (Baiersdorfer et al., 2019 (link)). All mRNAs were analyzed by agarose gel electrophoresis and were stored frozen at −20°C.
Mu Z., Wiehe K., Saunders K.O., Henderson R., Cain D.W., Parks R., Martik D., Mansouri K., Edwards R.J., Newman A., Lu X., Xia S.M., Eaton A., Bonsignori M., Montefiori D., Han Q., Venkatayogi S., Evangelous T., Wang Y., Rountree W., Korber B., Wagh K., Tam Y., Barbosa C., Alam S.M., Williams W.B., Tian M., Alt F.W., Pardi N., Weissman D, & Haynes B.F. (2022). mRNA-encoded HIV-1 Env trimer ferritin nanoparticles induce monoclonal antibodies that neutralize heterologous HIV-1 isolates in mice. Cell Reports, 38(11), 110514.
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10

mRNA Vaccine Manufacturing Process

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Antigens encoded by the mRNA vaccines were derived from the ancestral SARS-CoV-2 Wuhan-Hu-1 strain (GenBank MN908947.3). Nucleoside-modified mRNAs expressing SARS-CoV-2 full-length N (mRNA-N) or prefusion-stabilized S protein with two proline mutations (mRNA-S-2P) were synthesized by in vitro transcription using T7 RNA polymerase (MegaScript, Thermo Fisher Scientific) on linearized plasmid templates, as previously reported (26 (link)). Uridine triphosphate was replaced with one-methylpseudouridine (m1Ψ)-5′-triphosphate (TriLink, catalog no. N-1081) for producing nucleoside-modified mRNAs. Polyadenylated tails were added to the end of modified mRNAs for optimized protein expression. In vitro transcribed mRNAs were capped using ScriptCap m7G capping system and ScriptCap 2′-O-methyltransferase kit (ScriptCap, CellScript) (26 (link)), followed by purification using the cellulose purification method as previously described (27 (link)). Purified mRNAs were analyzed by agarose gel electrophoresis and were kept frozen at −20°C. The mRNAs were formulated into LNPs using an ethanolic lipid mixture of ionizable cationic lipid and an aqueous buffer system, as previously reported (28 (link), 57 (link)). Formulated mRNA-LNPs were prepared according to RNA concentrations (1 μg/μl) and were stored at −80°C for animal immunizations.
Hajnik R.L., Plante J.A., Liang Y., Alameh M.G., Tang J., Bonam S.R., Zhong C., Adam A., Scharton D., Rafael G.H., Liu Y., Hazell N.C., Sun J., Soong L., Shi P.Y., Wang T., Walker D.H., Sun J., Weissman D., Weaver S.C., Plante K.S, & Hu H. (2022). Dual spike and nucleocapsid mRNA vaccination confer protection against SARS-CoV-2 Omicron and Delta variants in preclinical models. Science translational medicine, 14(662), eabq1945.
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