One-Step Capping and 2´-O-Methylation (M0366)

Introduction

This protocol is designed to complete both capping and 2´-O-methylation in a single step. It involves incubating uncapped RNA with the Vaccinia Capping Enzyme (NEB #M2080, not included) and mRNA Cap 2´-O-Methyltransferase in the presence of GTP and SAM. The Vaccinia Capping Enzyme adds the cap at the 5´ end of the RNA followed by 2´-Omethylation by the methyltransferase. This protocol can synthesize up to 10 μg of cap-1 RNA in a 20 μl reaction. Reaction size can be scaled up as needed.


Protocol

Combine uncapped RNA and nuclease-free water in a final volume of 14.0 μl. (Refer to step 1 in the notes on use).

  1. Heat at 65°C for 5 minutes (Refer to step 2 in the notes on use).

  2. Place tube on ice for 5 minutes.

  3. Add the following components in the order specified:
    Denatured capped RNA (from above) 14.0 μl
    10X Capping Buffer 2.0 μl
    GTP (10 mM) 1.0 μl
    SAM (4 mM, dilute 32 mM stock to 4 mM) 1.0 μl
    Vaccinia Capping Enzyme (10 U/μl) 1.0 μl
    mRNA Cap 2´-O-Methyltransferase (50 U/μl) 1.0 μl
      20.0 μl

    Note: Use of RNase Inhibitor is recommended to enhance stability of RNA in the reaction. Add 0.5 μl of RNase Inhibitor (e.g., Murine RNase Inhibitor NEB #M0314) during reaction set up. Subtract the additional volume from the amount of H2O used in the reaction.

  4. Incubate at 37°C for 60 minutes (For RNA less than 200 nt long increase incubation time to 2 hours).

  5. Proceed with purification of the RNA (if required) for downstream applications.