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

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

The INCOGNITO™ T7 ARCA 5mC- & -RNA Transcription Kit* is optimized for high-yield synthesis of5-methylcytosine- pseudouridine-containing RNA (5mC -RNA) ARCA-capped RNA by in vitro transcription (IVT) of a DNA template that has a properly-oriented phage T7 RNA polymerase promoter usingT7-Scribe™ RNA polymerase, the canonical nucleotides ATP & GTP, the modified nucleotides 5-methylcytosine-5'-triphosphate (5mCTP) & pseudouridine-5'-triphosphate ( TP) and the anti-reverse cap analog(ARCA) m27,3'-OG[5']ppp[5']. It has been shown that 5mC -mRNAs are translated into protein at higherlevels and induce lower innate immune responses in human and other mammalian cells that expressvarious RNA sensors compared t°Corresponding unmodified mRNAs.1-4. Because ARCA contains a 3'-O-methyl group on the m7G nucleotide (Figure 1), ARCA can only beincorporated in the correct orientation at the 5' end of the RNA during an in vitro transcription/cappingreaction.5-7 This is not true for the standard cap analog (m7G[5']ppp[5']G). Thus, ARCA incorporationresults in the synthesis of capped RNA that is more efficiently translated in vivo than standard cap analog. Although yield varies with the DNA template and other factors, a standard 20 μl IVT reaction using 1 μg ofa control template DNA yields approximately 40 μg of 5mC -ARCA-capped RNA in 3 hours at 37°C. The5mC -ARCA-capped RNA can be enzymatically converted to a Cap 1 and 3'-poly(A)-tailed usingCELLSCRIPT's ScriptCap™ 2'-O-Methyltransferase and A-Plus™ Poly(A) Polymerase to generate5mC -mRNA for expression in cells. The ARCA/GA 5mC PreMix contains all four ribonucleotides (GA 5mC) and the ARCA. The PreMixensures the optimal concentration of each NTP and ratio of ARCA to GTP (4:1), maximizing transcriptcapping (~80%) and yield. Because the concentration of GTP in the reaction is limiting, the ARCA ispreferentially incorporated as the first or 5'-terminal G of the transcript.

RNase-Free DNase I is provided in a 50% glycerol solution containing 50 mM Tris-HCl, pH 7.5,10 mM CaCl2, 10 mM MgCl2 and 0.1% Triton® X-100. ScriptGuard RNase Inhibitor is supplied in a50% glycerol solution containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM DTT, 0.1 mM EDTAand 0.1% Triton® X-100. All other enzymes are provided in a 50% glycerol solution containing 50 mMTris-HCl, pH 7.5, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA and 0.1% Triton X-100.

Unit Definitions One unit of RNase-Free DNase I digests 1 µg of pUC19 DNA to oligodeoxynucleotides in 10 minutesat 37°C. One unit of ScriptGuard RNase Inhibitor results in 50% inhibition of 5 ng of RNase A. Activity ismeasured by the inhibition of hydrolysis of cyclic 2',3'-CMP by RNase A. Functional Testing The INCOGNITO T7 ARCA 5mC- & Ψ-RNA Transcription Kit is functionally tested under standardreaction conditions using the T7 Control Template DNA. The kit must produce at least 35 µg of RNAfrom 1 µg of the T7 Control Template DNA in 3 hours at 37°C. Contaminating Activity Assays All components of the INCOGNITO T7 ARCA 5mC- & Ψ-RNA Transcription Kit are free of detectableRNase and DNase activity, except for the inherent activity of the RNase-Free DNase I component.