The INCOGNITO™ T7 ARCA 5mC- & Ψ-RNA Transcription Kit is optimized for high-yield synthesis of 5-methyl-cytidine- pseudouridine-containing anti-reverse cap analog (ARCA)-capped RNA (ARCA-5mCΨ-RNA) from an in vitro transcription (IVT) reaction. A 3 hour, 20 μl reaction yields up to 40 μg of ARCA-5mCΨ-RNA from 1 μg of the 1.4 kb standard T7 control template DNA. Because ARCA contains a 3'-O-methyl group on the m7G nucleotide, ARCA can only be incorporated in the correct orientation at the 5' end of the RNA during an in vitro transcription/capping reaction.^1-3 This is not true for the standard cap analog. Thus, ARCA incorporation results in the synthesis of capped RNA that is more efficiently translated in vivo than standard cap analog.

The ARCA / GAΨ5mC PreMix contains all four ribonucleotides (GAΨ5mC) 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. Because the concentration of GTP in the reaction is limiting, the ARCA is preferentially incorporated as the first or 5'-terminal G of the transcript. Such RNA has been used in the landmark publication by Warren et al. (2010) to successfully reprogram multiple human cell types into induced pluripotent stem cells.⁴

It has been shown that Ψ-mRNAs and 5mC-mRNAs are translated into protein at higher levels and induce lower innate immune responses in human and other mammalian cells that express various RNA sensors compared to corresponding unmodified mRNAs.^5-8

INCOGNITO™ ARCA-5mCΨ-RNA can be further processed into 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 CELLSCRIPT™'s A-Plus™ Poly(A) Polymerase Tailing Kit (available separately).

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

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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• Manual

• SDS

These are both links to the manufacturer's page

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1. Stepinski, J. et al., (2001) RNA 7, 1486.
2. Peng, Z-H et al., (2002) Org. Lett. 4, 161.
3. Jemielity, J. et al., (2003) RNA 9, 1108.
4. Warren, L. et al., (2010) Cell Stem Cell 7, 618.
5. Karikó, K. et al., (2008) Molecular Therapy 16, 1833.
6. Anderson, B.R. et al., (2010) Nucl. Acids Res. 17, 5884.
7. Karikó, K. et al., (2005) Immunity 23, 165.
8. Karikó, K. and Weissman, D. (2007) Curr. Opin. Drug Discov. Devel. 10, 523.

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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.

Important

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

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200

• Co-transcriptionally cap 5-methyl-cytidine- pseudouridine-containing, in vitro transcribed RNA with Anti-Reverse cap analog (ARCA).
• Produce up to 40 µg of capped 5mCΨ-RNA from 1 µg of template in 3 hours.
• Optimized for maximum transcript capping and yield.
• Induce lower innate immune responses in mammalian cells by using 5mCΨ-mRNAs.

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200