ScriptCap™ Cap 1 Capping System

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

The ScriptCap™ Cap 1 Capping System provides both ScriptCap Capping Enzyme and ScriptCap 2'-OMethyltransferase allowing the user to produce either a Cap 0 or Cap 1 cap structure on the 5' end ofin vitro transcribed (IVT) RNA. ScriptCap Capping Enzyme catalyzes in vitro addition of a cap nucleotideto the 5' terminus of primary RNA that has a 5'-triphosphate group. A "cap nucleotide" or “cap” is aguanine nucleoside that is joined via its 5' carbon to a triphosphate group that is, in turn, joined to the5' carbon of the most 5' nucleotide of the primary mRNA transcript, and in most eukaryotes, the nitrogenat the 7 position of the guanine in the cap nucleotide is methylated. Such a capped transcript can berepresented as: m7G(5')ppp(5')N1(pN)x-OH(3') where m7G represents the N7-methylguanosine cap nucleoside, ppp represents the triphosphate bridgebetween the 5' carbons of the cap nucleoside and the first nucleotide of the primary RNA transcript, andN1(pN)x-OH(3') represents the primary RNA transcript, of which N1 is the most 5' nucleotide. The ScriptCap Capping Enzyme sequentially catalyzes three different enzymatic reactions: (1) RNA triphosphatase cleaves the 5' triphosphate of RNA to a diphosphate. pppN1(p)Nx-OH(3') ppN1(pN)x-OH(3') + Pi (2) RNA guanyltransferase joins GTP to the 5' diphosphate of the most 5' nucleotide (N1) of the RNA. ppN1(pN)x-OH(3') + GTP G(5')ppp(5')N1(pN)x-OH(3') + PPi (3) Guanine-7-methyltransferase, using S-adenosyl-methionine (SAM or AdoMet) as a co-factor,catalyzes methylation of the 7-nitrogen of guanine in the cap nucleotide. G(5')ppp(5')N1(pN)x-OH(3') + AdoMet m7G(5')ppp(5')N1(pN)x-OH(3') + AdoHyc This process, referred to as “capping,” improves the stability and translation efficiency of the RNAcompared to uncapped RNA. The capped RNA product built by ScriptCap Capping Enzyme has a“cap 0” structure. Cap 0 RNA can be converted to a "cap 1" structure in vitro by the simultaneous use ofScriptCap 2'-O-Methyltransferase in the capping reaction together with the ScriptCap Capping Enzyme. ScriptCap 2'-O-Methyltransferase prepares Cap 1-RNA from Cap 0-RNA by transferring a methyl groupfrom the donor molecule SAM to the 2'-O position of the penultimate nucleotide of a cap 0 RNA tosynthesize RNA with a cap 1 structure. m7GpppN1(pN)x-OH(3') + AdoMet m7Gppp[m2’-O]N1(pN)x-OH(3') + AdoHyc Cap 0 RNA Cap 1 RNA Cap 1 RNA can further increase in vivo translation efficiency of the mRNA as well as to help mark themRNA as "self RNA" relative to intracellular immuno-surveillance mechanisms. A standard ScriptCap Cap 1 Capping System reaction will cap approximately 60 µg of RNA, but reactionscan be scaled up or down to accommodate the user’s needs. Capped RNA from an ScriptCap Cap 1 Capping System reaction can be added directly to an A-Plus™Poly(A) Polymerase reaction (CELLSCRIPT), without prior cleanup, for poly(A)-tailing of the 3' ends of thecapped RNA allowing for convenient synthesis of mRNA. The ScriptCap Cap 1 Capping System offers an alternative to making capped RNA by co-transcriptionalcapping during an IVT reaction in which a dinucleotide cap analog (e.g., m7GpppG) is included in place ofa portion of the GTP.3 Provided that the 5' terminus of the RNA is not structured, the capping efficiencyusing the ScriptCap Cap 1 Capping System can approach 100%. In contrast, since the cap analogcompetes with GTP for initiation of transcription by the RNA polymerase, co-transcriptional cappingefficiency is limited by the concentration of the cap analog and the ratio of its concentration to that of theGTP. Thus, the percentage of RNA that is capped using a cap analog in a transcription reaction isalways less than 100%.4,5 The amount of capped RNA that can be made in a co-transcriptionalcapping reaction using a cap analog is also limited by the need to reduce GTP concentrations to permitthe cap analog t°Compete for initiation of transcription. On the other hand, co-transcriptional cappingwith a cap analog can be beneficial if the RNA to be capped has a highly structured 5' terminus. ContactCELLSCRIPT to discuss the options for your project. The ScriptCap Cap 1 Capping System improves upon co-transcriptional capping methods by ensuringvirtually 100% transcript capping, all caps in the proper orientation and the ability to produce largeamounts of capped RNA at a reasonable cost.

ScriptCap Capping Enzyme and ScriptCap 2'-O-Methyltransferase are provided in a 50% glycerol solution containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM Dithiothreitol (DTT), 0.1 mM EDTA and 0.1% Triton X-100®. ScriptGuard RNase Inhibitor is provided in a 50% glycerol solution containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM DTT, 0.1 mM EDTA and 0.1% Triton X-100®.

Unit Definitions One ScriptCap Cap 1 Capping System reaction produces 60 µg of 5'-Cap 0 or Cap 1 capped RNA. One unit of ScriptCap Capping Enzyme releases 1 nmole of inorganic phosphate from GTP in10 minutes at 37°C under standard assay conditions. One unit of ScriptCap 2'-O-Methyltransferase methylates one picomole of a control Cap 0 RNA in1 hour at 37°C under standard assay conditions. One unit of ScriptGuard RNase Inhibitor results in 50% inhibition of 5 ng of RNase A. Contaminating Activity Assays All components of the ScriptCap Cap 1 Capping System are free of detectable RNase and DNaseactivities.