ScriptCap™ 2′-O-Methyltransferase Kit

Achieve ~100% Cap 1 capping of Cap-0 RNA from any source, a level unattainable using cap analogs.
Enhance mRNA stability and translation efficiency through post-transcriptional 5’ capping.
Use simultaneously with ScriptCap™ m⁷G Capping System to produce Cap 1 caps in a single reaction.
Simplified workflow with direct input of capped RNA into A-Plus™ Poly(A) Polymerase tailing reactions with no cleanup required.

Product is for research use only (RUO)

Product Description:

The ScriptCap™ 2′-O-Methyltransferase Kit provides conversion of Cap 0-RNA from any source to Cap 1-RNA with ~100% efficiency, a level that cannot be obtained using co-transcriptional capping methods. 5’ Capping enhances mRNA stability and translation efficiency in cells compared to uncapped RNA, with Cap 1 methylation further boosting in vivo translation efficiency. A standard reaction methylates 60 μg of Cap 0-RNA and can be scaled up or down to accommodate user needs.

Cap 0-RNA can be produced by enzymatic capping using the ScriptCap™ m⁷G Capping System or by in vitro transcription of a DNA template in the presence of a dinucleotide cap analog (e.g., using MessageMAX™ T7 ARCA-Capped Message Transcription Kit V2). Uncapped RNA can be converted to Cap 1-RNA in a single reaction by combining the ScriptCap™ m⁷G Capping System and 2’-O-Methyltransferase.

ScriptCap™ capped RNA can be added directly to A-Plus™ Poly(A) Polymerase tailing reactions without cleanup for seamless synthesis of fully 5’ capped and 3’ tailed mRNA.

Materials Supplied:

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

ScriptCap™ 2'-O-Methyltransferase Kit Contents (25 reactions)
Kit Component	Reagent Volume
ScriptCap™ 2'-O-Methyltransferase, 100 U/μl in 50% glycerol, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT), 0.1 mM EDTA and 0.1% Triton® X-100	100 μl
10X ScriptCap™ Capping Buffer 0.5 M Tris-HCl, pH 8.0, 60 mM KCl and 12.5 mM MgCl₂	250 μl
20 mM S-adenosyl-methionine (SAM)	65 μl
ScriptGuard™ RNase Inhibitor, 40 U/μl in 50% glycerol, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM DTT, 0.1 mM EDTA and 0.1% Triton X-100	65 μl
RNase-Free Water	2 x 1.4 ml

Materials Required, but not Supplied

Cap 0 RNA substrate
Materials or kits for purification of the RNA product

Terms & Conditions: The Product listed is currently offered by CELLSCRIPT™ for research use only under the defined Terms and Conditions.

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Troubleshooting Guide:

Symptom	Solution
Low capping efficiency or 2'-O-methylation efficiency	RNA to be treated with ScriptCap™ 2'-O-Methyltransferase should be purified and resuspended in RNase-free water. Do not resuspend the RNA in an EDTA-containing solution.
	Prior to 2'-O-methylation, purify the input RNA using a method that removes residual proteins, contaminants and unincorporated nucleotides and/or cap analogs.
	Verify that ScriptGuard™ RNase Inhibitor was added to the reaction.
	SAM slowly degrades at room temperature and above. Keep SAM solutions on ice at all times.
	Increase the reaction incubation time. For example, up to 3 hours at 37^oC.

	Some RNAs form stable structures (e.g., homodimers, hairpins) at the 5' end, limiting access by Capping Enzyme or 2'-O-Methyltransferase. Analyze the sequence and increase the RNA denaturation temperature to above the T_m (e.g., to 65^oC for 20 min, 75^oC for 10 min, 85^oC for 5 min). If the 5' end is highly structured, it might be necessary to modify the 5' end sequence using molecular biology techniques. Often this can be accomplished by making a single point mutation within the first 5 bases of the DNA template for the RNA transcript (non-coding region).
White precipitate in reaction buffer	Incubate the reaction buffer at 37°C for 5 minutes then mix thoroughly to dissolve the precipitate.
White precipitate in reaction buffer	Do not store the kit at –70°C.