• My NEB
  • Print
  • PDF
  • HEN1 miRNA Methyltransferase

    Description

    HEN1 is a plant miRNA Methyltransferase that methylates the terminal ribose in short double-stranded RNAs. In Arabidopsis, 2´-0-methylation of the 3´ ribose of miRNAs and siRNAs is mediated by HEN1 (1, 2). In HEN1 mutants, small RNAs were found unmethylated with a 3´ poly (U) tail, suggesting 3´ end methylation protects small RNAs from uridylation (3). Purified HEN1 methylates both miRNA/miRNA and siRNA/siRNA duplexes with a preference for 21-24 nucleotide RNA duplexes with 2 nucleotide overhangs (4). HEN1 does not methylate single-stranded RNA or DNA.

    Product Source

    An E. coli strain that carries the cloned HEN1 from Arabidopsis.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 2-2010X
    S-adenosylmethionine (SAM)-2032 mM

    Advantages and Features

    Applications

    • 3´ end labeling of siRNA and miRNA

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to transfer 1 pmol of methyl group to a miRNA duplex in 10 minutes at 37°C.

    Reaction Conditions

    1X NEBuffer 2
    Supplement with 128 μM S-adenosylmethionine (SAM)
    Incubate at 37°C

    1X NEBuffer 2:
    50 mM NaCl
    10 mM Tris-HCl
    10 mM MgCl2
    1 mM DTT
    pH 7.9 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    100 mM NaCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    pH 7.5 @ 37°C

    Heat Inactivation

    70°C for 10 min

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • RNase Activity (1 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 1 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    Notes

    1. Up to 500 ng miRNA duplex or 75 pmol 3´ end can be fully methylated in a 25 μl reaction.
    2. SAM should be freshly diluted 1:10 with H2O prior to use.
    3. For miRNA isotope labeling, use 2 μl [14C] SAM instead of cold SAM; miRNA amount can be reduced to increase specific activity.
    4. S-adenosylmethionine is stored at –20°C as 32 mM solution dissolved in sulfuric acid (0.005 M) and 10% ethanol. SAM in this solution stored under ideal conditions remains active for up to 6 months. SAM is unstable at (pH 7.5), 37°C, and should be replenished for reactions incubated longer than 4 hours. Many problems in achieving complete digestion can be alleviated by using fresh SAM.

    References

    1. Park, W., et al. (2002). Curr. Biol.. 12, 1484-1495.
    2. Yu, B., et al. (2005). Science . 307, 932-935.
    3. Li, J., et al. (2005). Curr. Biol. . 15, 1501-1507.
    4. Yang, Z., et al. (2006). Nucleic Acids Res. 34, 667-675.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. Protocol for a Typical miRNA Methylation Reaction (M0228)