The ScriptCap™ Cap 1 Capping System provides both ScriptCap Capping Enzyme and ScriptCap 2'-O-Methyltransferase allowing the user to produce either a Cap 0 or Cap 1 cap structure on the 5' end of in vitro transcribed (IVT) RNA. ScriptCap Capping Enzyme catalyzes in vitro addition of a cap nucleotide to the 5' terminus of primary RNA that has a 5'-triphosphate group. A "cap nucleotide" or “cap” is a guanine nucleoside that is joined via its 5' carbon to a triphosphate group that is, in turn, joined to the 5' carbon of the most 5' nucleotide of the primary mRNA transcript, and in most ukaryotes, the nitrogen at the 7 position of the guanine in the cap nucleotide is methylated. Such a capped transcript can be represented as:
m7G(5')ppp(5')N1(pN)x-OH(3')
where m7G represents the N7-methylguanosine cap nucleoside, ppp represents the triphosphate bridge between the 5' carbons of the cap nucleoside and the first nucleotide of the primary RNA transcript, and N1(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 RNA compared 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 of ScriptCap 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 group from the donor molecule SAM to the 2'-O position of the penultimate nucleotide of a cap 0 RNA to synthesize 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 the mRNA as "self RNA" relative to intracellular immuno-surveillance mechanisms.1,2
A standard ScriptCap Cap 1 Capping System reaction will cap approximately 60 μg of RNA, but reactions can 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 the capped RNA allowing for convenient synthesis of mRNA. The ScriptCap Cap 1 Capping System offers an alternative to making capped RNA by co-transcriptional capping during an IVT reaction in which a dinucleotide cap analog (e.g., m7GpppG) is included in place of a portion of the GTP.3 Provided that the 5' terminus of the RNA is not structured, the capping efficiency using the ScriptCap Cap 1 Capping System can approach 100%. In contrast, 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 its concentration to that of the GTP. Thus, the percentage of RNA that is capped using a cap analog in a transcription reaction is always less than 100%.4,5 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 GTP concentrations to permit the cap analog to 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 highly structured 5' terminus. Contact CELLSCRIPT to discuss the options for your project.
The ScriptCap Cap 1 Capping System improves upon co-transcriptional capping methods by ensuring virtually 100% transcript capping, all caps in the proper orientation and the ability to produce large amounts of capped RNA at a reasonable cost.
REFERENCES
1. Kuge, H. et al., (1998) Nucl. Acids Res. 26, 3208.
2. Schlee, M. and Hartmann, G., (2016) Nat. Rev. Immunol. 16, 566.
3. Konarska, M.M. et al., (1984) Cell 38, 731.
4. Jemielity, J. et al., (2003) RNA 9, 1108.
5. Grudzien, E. et al., (2004) RNA 10, 1479.
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