INCOGNITO™ T7 mScript™ Ψ-mRNA Production System

All-in-one kit to produce pseudouridine-containing, fully 5’ capped and 3’ tailed mRNA (Ψ-mRNA).
Combine the benefits of high-yield transcription with post-transcriptional capping and tailing.
Produce large amounts of Ψ-mRNA with virtually 100% transcript capping.
Create variable 3’ poly(A) tail lengths, from 50 to >300 A’s.
Lower immunogenicity in mammalian cells through incorporation of Ψ in synthesized mRNA.

Product is for research use only (RUO).

Use and Label Licenses for Specific Products: Purchaser receives a limited non-exclusive, non-transferable right to use the Products purchased from CELLSCRIPT™ solely for its own internal laboratory research use. See the Label License tab for more information.

Product Description:

The INCOGNITO™ T7 mScript™ Ψ-mRNA Production System provides all enzymes and reagents for making low immunogenicity pseudouridine (Ψ)-containing, 5'-capped and 3'-polyadenylated mRNA. The kit includes reagents for four workflow modules, (1) high-yield in vitro transcription of linear double-stranded DNA templates using the T7 mScript™ Enzyme Solution, the canonical nucleotides ATP, CTP, GTP and the modified nucleotide pseudouridine-5'-triphosphate (ΨTP), (2) enzymatic capping of the RNA using the ScriptCap™ Cap 1 Capping System (contains both ScriptCap™ Capping Enzyme and 2'-O-Methyltransferase) for making mRNA with a Cap 1 cap structure, (3) A-Plus™ Poly(A) Polymerase for adding a 3'-poly(A) tail and (4) 5 M NH₄OAc as a convenient RNA/mRNA purification method.

Post-transfection, capped and tailed mRNA has increased stability and translation efficiency in most eukaryotic cell lines. Additionally, it has been shown that Ψ-mRNAs are translated into protein at higher levels and induce lower innate immune responses in humans and other mammalian cells that express various RNA sensors compared to corresponding unmodified mRNAs. The INCOGNITO™ mScript™ System generates modified mRNA with virtually 100% transcript capping and user-defined poly(A) tail length. Poly(A) tail lengths can be generated much longer than is possible using a template-encoded tail, even greater than 300 A’s. This mRNA is suitable for use in transfection and microinjection experiments as well as in vitro translation systems.

For maximum reduction of immunogenicity, combine INCOGNITO™ Ψ-mRNA with the Min-Immune™ Gold dsRNA Removal Kit to produce virtually dsRNA-free, ultra-low immunogenicity mRNA that is suitable for downstream applications, such as cell and gene therapy research and mRNA vaccine development.

Materials Supplied:

Important Store at -20°C in a freezer without a defrost cycle. Do not store at –70°C.

INCOGNITO™ T7 mScript™ Ψ-mRNA Production System Kit Contents \| 25 Reactions. (Module 1 of 4)
Kit Module	Kit Component	Reagent Volume
In Vitro Transcription	T7 mScript™ Enzyme Solution	50 µl
	10X T7 mScript™ Transcription Buffer II	50 µl
	100 mM Dithiothreitol (DTT)	50 µl
	ΨTP PreMix 25 mM each GTP, ATP, ΨTP, CTP	180 µl
	RNase-Free DNase I, 1 U/μl	25 µl

INCOGNITO™ T7 mScript™ Ψ-mRNA Production System Kit Contents \| 25 Reactions. (Module 2 of 4)
Kit Module	Kit Component	Reagent Volume
Post-Transcriptional Capping	ScriptCap™ Capping Enzyme, 10 U/μl	100 µl
	ScriptCap™ 2'-O-Methyltransferase, 100 U/μl	100 µl
	10X ScriptCap™ Capping Buffer 0.5 M Tris-HCl (pH 8.0), 60 mM KCl and 12.5 mM MgCl₂	250 µl
	20 mM S-adenosyl-methionine (SAM)	125 µl
	20 mM GTP	125 µl

INCOGNITO™ T7 mScript™ Ψ-mRNA Production System Kit Contents \| 25 Reactions. (Module 3 of 4)
Kit Module	Kit Component	Reagent Volume
Poly(A) Tailing	A-Plus™ Poly(A) Polymerase, 4 U/µl	130 µl
	10X A-Plus™ Poly(A) Tailing Buffer 0.5 M Tris-HCl (pH 8.0), 2.5 M NaCl and 100 mM MgCl₂	300 µl
	20 mM ATP	150 µl

INCOGNITO™ T7 mScript™ Ψ-mRNA Production System Kit Contents \| 25 Reactions. (Module 4 of 4)
Kit Module	Kit Component	Reagent Volume
Common Usage	ScriptGuard™ RNase Inhibitor, 40 U/µl	90 µl
	RNase-Free Water	12 ml
	5 M Ammonium Acetate	12 ml

T7 Control Template DNA: Is a linearized 4.1 kb plasmid that contains a T7 promoter followed by a phage lambda dsDNA insert that encodes a 1,375 base runoff transcript. The Control Template DNA is provided at a concentration of 0.5 µg/µl in T₁₀E₁ Buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA).

Materials Required, but not Supplied

A DNA template for transcription of your RNA of interest
Materials or kits for purification of the RNA product
RNase-free TE Buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA)
Optional: TE saturated phenol/chloroform, 0.5-1 M EDTA

Terms and Conditions: The Product listed is currently offered by CELLSCRIPT™ for research use only under the defined Terms and Conditions.

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FAQs: For answers to Frequently Asked Questions about our products, visit the FAQ library.

Still have questions? Our mRNA experts are here to help.

Troubleshooting Guide:

Synthesis of IVT RNA
Symptom	Solution
Low yields or less than full-length transcripts	Cleanup the templates to remove any RNase or other contaminants.
	Verify that ScriptGuard™ RNase Inhibitor was added to the reaction.
	Extend the incubation time. Do not extend the reaction time beyond 3 hours.
	Increase the template concentration.
	Increase the reaction temperature to 42°C.
Assembled reaction formed an insoluble precipitate	Repeat assembly of the reaction at >22°C.
White precipitate in reaction buffer	Incubate the reaction buffer at 37°C for 5 minutes then mix thoroughly to dissolve the precipitate.
White precipitate in reaction buffer	Do not store the kit at –70°C.

Synthesis of Capped RNA
Symptom	Solution
Low capping efficiency	Cleanup the templates to remove any RNase or other contaminants.
	Verify that ScriptGuard™ RNase Inhibitor was added to the reaction.
	SAM slowly degrades at room temperature and above. Keep SAM solutions on ice at all times.
	Increase the capping reaction incubation time. For example, up to 3 hours at 37°C.
	Some RNAs form stable structures (e.g., homodimers, hairpins) at the 5' end, limiting access by Capping Enzyme or 2'-O-Methyltransferase. Analyze the sequence and increase the RNA denaturation temperature to above the T_m (e.g., to 65°C for 20 min, 75°C for 10 min, 85°C for 5 min). If the 5' end is highly structured, it might be necessary to modify the 5' end sequence using molecular biology techniques. Often this can be accomplished by making a single point mutation within the first 5 bases of the DNA template for the RNA transcript (non-coding region). Contact CELLSCRIPT™ Technical Support for suggestions and recommendations.
White precipitate in reaction buffer	Incubate the reaction buffer at 37°C for 5 minutes then mix thoroughly to dissolve the precipitate.
White precipitate in reaction buffer	Do not store the kit at –70°C.

Synthesis of Poly(A)-Tailed RNA
Symptom	Solution
Poly(A) tails are longer than expected	Decrease the time of incubation of the reaction.
Poly(A) tails are longer than expected	Decrease the amount of A-Plus™ Poly(A) Polymerase used in the reaction.
Poly(A) tails are shorter than expected	Increase the time of incubation of the reaction.
Poly(A) tails are shorter than expected	Increase the amount of A-Plus™ Poly(A) Polymerase used in the reaction.
No poly(A) tails are observed	Enzyme is inactive. Store only at –20°C. Keep on ice when not in the freezer.
No poly(A) tails are observed	ATP is hydrolyzed. Do not expose to elevated temperatures.

Uses and Label Licenses for Specific Products

INCOGNITO™ kits or other products or services comprising N1-methyl-pseudouridine, pseudouridine, 5-methyl-cytidine and/or other modified nucleotides for use in making RNA or mRNA (the “Products”) that are covered by technologies and IP disclosed in U.S. Patent Nos. 8,278,036 and 9,750,824 and PCT Publ. Nos. WO 2007/024708 and WO/2011/071931 and U.S. and international divisional, continuation, or other patents and patent applications derived therefrom that are owned by the University of Pennsylvania and licensed to CELLSCRIPT™ (“Products”) are sold for research use only.

By purchasing these Products from CELLSCRIPT™ or an authorized distributor of CELLSCRIPT™, Purchaser receives a limited non-exclusive, non-transferable right to use the Products purchased from CELLSCRIPT™ solely for its own internal laboratory research use (the “Product Use”). Such Product Use expressly excludes any Commercial Use comprising [A] clinical or other use in humans, [B] veterinary, livestock, agricultural or commercial use in animals, and/or [C] manufacture, distribution, importation, exportation, or sale of any products and/or services made using Products, it being understood that [A]–[C] are separately and/or collectively defined as a “Commercial Use” herein. By purchase of Products, Purchaser agrees not to make, import, use, transfer, offer for sale, or sell Products, components of Products, or derivatives of Products, including modified nucleoside-containing RNA or cells made by use of Products, or to provide services, information, or data obtained by the use thereof in exchange for money or other consideration. CELLSCRIPT™ provides no warranties (statutory or implied) concerning non-infringement of intellectual property rights of any other parties, and all such warranties are expressly disclaimed. Please contact CELLSCRIPT™ to inquire about a license for a Commercial Use.

The license remains in effect as long as the product is being used in accordance with the license terms. The license is terminated if the user breaches any of the license terms, such as using the product for commercial purposes without proper authorization.