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HEN1 miRNA Methyltransferase |
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Prices are in US dollars and valid only for US orders.
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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.
Source:
An E. coli strain that carries the cloned HEN1 from Arabidopsis.
Applications:- 3´ end labeling of siRNA and miRNA
Reagents Supplied:
NEBuffer 2 (10X)
S-adenosylmethionine (SAM) (32 mM)
Enzyme Properties
Heat Inactivation:
70°C for 10 minutes
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 2
Supplemented with 128 μM S-adenosylmethionine (SAM) and 500 ng miRNA duplex
Incubate at
37°C.
1X NEBuffer 2:
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.9 @ 25°C
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.
Concentration:
50,000 units/ml
Storage Conditions:
10 mM Tris-HCl
100 mM NaCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
pH 7.5 @ 37°C
Storage Temperature:
-20°C
Notes
General notes:- 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.
- Up to 500 ng miRNA duplex or 75 pmol 3´ end can be fully methylated in a 25 μl reaction.
- SAM should be freshly diluted 1:10 with H2O prior to use.
- For miRNA isotope labeling, use 2 μl [14C] SAM instead of cold SAM; miRNA amount can be reduced to increase specific activity.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 50 units of HEN1 miRNA Methyltransferase with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.5% of the total radioactivity.
Endonuclease Activity::
Incubation of a 10 μl reaction containing 50 units of HEN1 miRNA Methyltransferase with 300 ng pUC19 plasmid DNA for 4 hours at 37°C resulted in <10% conversion from supercoiled to nicked molecules as determined by agarose gel electrophoresis.
RNase Assay::
Incubation of a 10 μl reaction containing 50 units of HEN1 miRNA Methyltransferase with 40 ng RNA (a 0.3 kb in vitro transcript) for 2 hours at 37°C, resulted in no detectable degredation of the RNA as determined by denaturing PAGE analysis.
References
- Park, W., et al. (2002) Curr. Biol., 12, 1484-1495.
- Yu, B., et al. (2005) Science, 307, 932-935.
- Li, J., et al. (2005) Curr. Biol., 15, 1501-1507.
- Yang, Z., et al. (2006) Nucleic Acids Res., 34, 667-675.
Reagents Sold Separately
NEBuffer 2
S-adenosylmethionine (SAM)
Companion Products
RNase Inhibitor
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