The INCOGNITO™ T7 5mC- & -RNA Transcription Kit* is optimized for high-yield synthesis of 5-methylcytosine- pseudouridine-containing RNA (5mC -RNA) by in vitro transcription (IVT) of a DNA template that has a properly-oriented phage T7 RNA polymerase promoter using T7-Scribe™ RNA polymerase, the canonical nucleotides ATP & GTP, and the modified nucleotides 5-methylcytosine-5'-triphosphate (5mCTP) & pseudouridine-5' triphosphate ( TP). Although yield varies with the DNA template and other factors, a standard 20 μl IVT reaction using 1 μg of a control template DNA yields approximately 100 μg of 5mC -RNA in 2 hours at 37oC. The 5mC -RNA can be enzymatically 5'-capped and 3'-poly(A)-tailed using CELLSCRIPT's ScriptCap™ m7G Capping System, ScriptCap™ 2'-O-Methyltransferase and A-Plus™ Poly(A) Polymerase to generate 5mC -mRNA for expression in cells. It has been shown that 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.
The mission of CELLSCRIPT™ is to provide the best products and technologies for making and using RNA for translation in cells for clinical research and therapeutics.
Current products include kits for in vitro transcription, 5' RNA capping using either a cap analog or capping enzymes, and 3' RNA polyadenylation, as well as all-in-one kits for making capped, poly(A)-tailed mRNA for translation in cells.
CELLSCRIPT™ recently introduced INCOGNITO™ RNA Transcription Kits for in vitrosynthesis of RNA that contains modified nucleosides, such as pseudouridine (Ψ) and/or 5-methylcytidine (m5C) in place of the corresponding U or C canonical nucleosides (Figure 1). These kits are so-named because the capped, polyadenylated and nucleoside-modified RNA products (called "INCOGNITO mRNAs") are disguised so they do not induce innate immune responses to the same extent as the corresponding unmodified mRNAs when transfected into mammalian cells that express a variety of RNA sensors.