ScriptCap™ m7G Capping System

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

ScriptCap™ m7G Capping System catalyzes in vitro addition of a cap nucleotide to the 5' terminus ofprimary RNA that has a 5'-triphosphate group, such as RNA obtained from an in vitro transcriptionreaction. A "cap nucleotide" or “cap” is a guanine nucleoside that is joined via its 5' carbon to atriphosphate group that is, in turn, joined to the 5' carbon of the most 5' nucleotide of the primary mRNAtranscript, and in most eukaryotes, the nitrogen at the 7 position of guanine in the cap nucleotide ismethylated. Such a capped transcript can be represented as: m7G(5')ppp(5')N1(pN)x-OH(3') where m7G represents the 7-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 m7G Capping System 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 (AdoMet) as a co-factor, catalyzesmethylation 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 from a ScriptCap m7G Capping System has a“cap 0” structure. Cap 0 RNA can be converted to a "cap 1" structure in vitro by using CELLSCRIPT’sScriptCap 2'-O-Methyltransferase in the capping reaction together with the ScriptCap m7G CappingSystem. In addition to having a 7-methyl-G cap nucleotide (m7G), Cap 1 RNA also has a 2'-O-methylgroup on the 5'-penultimate (N1) nucleotide, which can further increase in vivo translation efficiency of themRNA. A standard ScriptCap m7G 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 m7G Capping System reaction can be added directly to a CELLSCRIPTA-Plus™ Poly(A) Polymerase reaction for poly(A)-tailing of the 3' ends of the capped RNA. The ScriptCap m7G Capping System offers an alternative to making capped RNA by co-transcriptionalcapping during an in vitro transcription (IVT) reaction in which a dinucleotide cap analog (e.g.,m7G(5')ppp(5')G) is included in place of a portion of the GTP.2 Provided that the 5' terminus of the RNA isnot structured, the capping efficiency using the ScriptCap m7G Capping System can approach 100%. Incontrast, since the cap analog competes with GTP for initiation of transcription by the RNA polymerase,co-transcriptional capping efficiency is limited by the concentration of the cap analog and the ratio of itsconcentration to that of the GTP. Thus, the percentage of RNA that is capped using a cap analog in atranscription reaction is always less than 100%.3,4 The amount of capped RNA that can be made in a co-transcriptional capping reaction using a cap analog is also limited by the need to reduce GTPconcentrations to permit the cap analog t°Compete for initiation of transcription. On the other hand,co-transcriptional capping with a cap analog can be beneficial if the RNA to be capped has a highlystructured 5' terminus. Contact CELLSCRIPT to discuss the options for your project.

Unit Definitions One ScriptCap m7G Capping System reaction produces 60 µg of 5'-Cap 0 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. Functional Testing The ScriptCap m7G Capping System is functionally tested for mRNA triphosphatase, guanylyltransferase and guanine-7-methyltransferase activities in multiple assays. Contaminating Activity Assays All components of the ScriptCap m7G Capping System are free of detectable RNase and DNaseactivities.