INCOGNITO™ T7 ARCA 5mC- & Ψ-RNA Transcription Kit

Co-transcriptionally cap 5-methyl-cytidine- and pseudouridine-containing IVT RNA with Anti-Reverse Cap Analog (ARCA).
Optimized for maximum transcript capping and yield.
Produce up to 40 µg of ARCA-5mCΨ-RNA from 1 µg of template in three hours.
Lower immunogenicity in mammalian cells through incorporation of Ψ and 5mC 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 ARCA 5mC- & Ψ-RNA Transcription Kit is optimized for high-yield synthesis of 5-methyl-cytidine- and pseudouridine-containing Anti-Reverse Cap Analog (ARCA)-capped RNA (ARCA-5mCΨ-RNA) from an in vitro transcription (IVT) reaction. A three hour, 20 μl reaction yields up to 40 μg of ARCA-5mCΨ-RNA from 1 μg of the control template DNA. ARCA ensures correct 5′ end incorporation, resulting in RNA that translates more efficiently in vivo than standard cap analogs.

The ARCA / GAΨ5mC PreMix contains all four ribonucleotides (canonical nucleotides GTP and ATP and modified nucleotides ΨTP and 5mCTP) and the ARCA. The PreMix ensures the optimal concentration of each NTP and ratio of ARCA to GTP (4:1), maximizing transcript capping (~80%) and yield.

Modified Ψ-mRNAs and 5mC-mRNAs translate into higher protein levels and lower innate immune responses in human and other mammalian cells that express various RNA sensors compared to unmodified mRNAs.

INCOGNITO™ ARCA-5mCΨ-RNA can be further processed into low immunogenicity mRNA for expression in cells through the use of post-transcriptional capping using CELLSCRIPT™'s ScriptCap™ 2'-O-Methyltransferase (for conversion to a Cap 1 structure) and post-transcriptional tailing using A-Plus™ Poly(A) Polymerase Tailing Kit (available separately).

For maximum reduction of immunogenicity, combine INCOGNITO™ ARCA-5mCΨ-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 ARCA 5mC- & Ψ-RNA Transcription Kit Contents (10 reactions)
Kit Component	Reagent Volume
T7-Scribe™ Enzyme Solution	20 μl
10X T7-Scribe™ Transcription Buffer	20 μl
ARCA / GAΨ5mC PreMix (18.75 mM ATP, ΨTP & 5mCTP; 3.75 mM GTP & 15 mM ARCA)	80 μl
20 mM GTP	20 μl
100 mM Dithiothreitol (DTT)	20 μl
RNase-Free DNase I, 1 U/μl	10 μl
ScriptGuard™ RNase Inhibitor	10 μl
T7 Control Template DNA, 0.5 μg/μl	10 μl
RNase-Free Water	1.4 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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→ CELLSCRIPT™ RNA 5′ Capping Comparison Table

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:

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 4 hours.
	Increase the template concentration.
	Increase the reaction temperature to 42^oC.
	Check for stable secondary structure (T_m >37^oC) in the DNA template which can cause T7 RNAP pausing or dissociation from the template.
Assembled reaction formed an insoluble precipitate	Repeat assembly of the reaction at >22^oC.
White precipitate in reaction buffer	Incubate the reaction buffer at 37^oC for 5 minutes then mix thoroughly to dissolve the precipitate.
White precipitate in reaction buffer	Do not store the kit at –70^oC.

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.