New England Biolabs, M0366S, mRNA Cap 2'-O-Methyltransferase

mRNA Cap 2´-O-Methyltransferase adds a methyl group at the 2´-O position of the first nucleotide adjacent to the cap structure at the 5´ end of the RNA. Related Categories RNA Capping Specification Unit Definition One unit is defined as the amount of enzyme required to methylate 10 pmoles of 80 nt long capped RNA transcript in 1 hour at 37°C. Reaction Conditions 1X Capping Buffer Supplement with 0.2 mM S-adenosylmethionine (SAM) Incubate at 37°C 1X Capping Buffer 50 mM Tris-HCl 5 mM KCl 1 mM MgCl2 1 mM DTT (pH 8 @ 25°C) Storage Buffer 20 mM Tris-HCl 100 mM NaCl 1 mM DTT 0.1 mM EDTA 50% Glycerol 0.1% (w/v) Triton® X-100 pH 8 @ 25°C FAQ Q: What kind of RNA can be methylated using the mRNA Cap 2'-O-Methyltransferase? A: mRNA Cap 2'-O-Methyltransferase requires capped RNA as substrate. Capped RNA can be prepared via in-vitro transcription (T7 High Yield RNA Synthesis Kit, NEB# E2040) using cap analog (ARCA 3´-O-Me-m7G(5')ppp(5')G RNA Cap Structure Analog, NEB# S1411) or by enzymatic capping using the Vaccinia capping enzyme (NEB# M2080). Q: I want to use the mRNA Cap 2'-O-Methyltransferase with the Vaccinia Capping System (NEB# M2080) in a one-step reaction to produce cap 1 RNA. Is that possible? A: Yes, mRNA Cap 2'-O-Methyltransferase can be simultaneously used with the Vaccinia capping enzyme in a one-step reaction. This involves capping and methylation of the RNA in the same reaction. The Vaccinia capping enzyme adds the m7G cap at the 5’ end of the RNA to produce capped RNA. The 2’-O-methyltransferase utilizes this RNA as a substrate and adds a methyl group at the 2’-O position of the first transcribed nucleotide to produce cap-1 RNA. Refer to the protocol section for reaction set up. Q: What precautions should be taken while using the mRNA Cap 2'-O-Methyltransferase? A: It is important to make sure that RNA being used in the reaction is free from salts and EDTA. It should preferably be re-suspended in nuclease-free water. We strongly recommend using gloves and nuclease-free tubes, pipette tips and reagents to prevent RNase contamination. RNase Inhibitor (NEB# M0314 ) may be used in the reaction to prevent degradation due to contaminating RNases. Q: What is Cap-0 and Cap-1? A: Cap-0 is a N7-methyl guanosine connected to the 5′ nucleotide through a 5′ to 5′ triphosphate linkage, typically refers to as m7G cap or m7Gppp- in the literature. In the cell, the Cap-0 structure is essential for efficient translation of the mRNA that carries the cap. An additional methylation on the 2′O position of the initiating nucleotide generates Cap-1, or refers to as m7GpppNm-, where Nm denotes any nucleotide with a 2′O methylation. Cap-1 has been shown to be important in evading the cellular innate immune response in vivo. Q: Can Vaccinia Capping Enzyme (NEB #M2080) use cap analogs in capping reaction? A: No. Vaccinia Capping Enzyme uses GTP and SAM to generate a cap structure on 5′ triphosphate RNA, typically a synthetic transcript generated by in vitro transcription. On the other hand, cap analogs, such as GpppG (NEB #S1407), m7GpppG (NEB #S1404), m7GpppA (NEB #S1405S) and anti-reverse cap analog (ARCA; NEB #S1411) are used during in vitro transcription, where the cap analog is directly incorporated into the transcript as the first nucleotide at the 5′ end.