Enzymes for Molecular Biology |
Phosphatases, Pyrophosphatases, and Kinases
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Applications
- Conversion of 5´-triphosphorylated RNA to 5´-monophosphorylated
RNA for use in 5´-RNA ligation-tagging methods using T4 RNA Ligase.
- Analysis of 5´-end structure of RNA.
- Preparation of substrate RNA molecules for subsequent degradation using
EPICENTRE's Terminator™ 5´-Phosphate-Dependent Exonuclease.
RNA 5´ Polyphosphatase* is a Mg2+-independent phosphohydrolase
discovered and characterized by EPICENTRE scientists. The enzyme sequentially
removes the γ and β phosphates from 5´-triphosphorylated RNA
(such as primary RNA transcripts):
5´ pppN—OH 3´ → 5´ pN—OH 3´ + 2 Pi
RNAs with a 5´-diphosphorylated end are also converted to 5´-monophosphorylated
RNA by RNA 5´ Polyphosphatase:
5´ ppN—OH 3´ → 5´ pN—OH 3´ + Pi
RNA Polyphosphatase has no activity on RNA with a 5´ cap (e.g., 5´ m7GpppN—OH
3´), or a 5´-monophosphorylated end (5´ pN—OH
3´). However, both NTPs and dNTPs are substrates for the enzyme,
yielding the corresponding NMPs and dNMPs + inorganic phosphate: (d)NTP → (d)NMP
+ 2Pi
Unit Definition: One unit of RNA 5´ Polyphosphatase releases
1 nmol of inorganic phosphate from ATP in 1 hour at 37°C under standard
assay conditions.
Storage Buffer: 50% glycerol containing 0.05 M Tris-HCl (pH 7.5), 0.1
mM EDTA, 1 mM DTT, 0.1 M NaCl, and 0.1% Triton® X-100.
10X Reaction Buffer: 0.5 M HEPES-KOH (pH 7.5), 1 M NaCl, 10 mM EDTA,
1% β-mercaptoethanol, and 0.1% Triton® X-100.
Inactivation/Inhibitors: RNA 5´ Polyphosphatase is inhibited
~50% by 100 mM of inorganic phosphate (Pi) using ATP as a substrate.
Quality Control: RNA 5´ Polyphosphatase is free of detectable
DNA exo- and endonuclease, and RNase activities.
*Covered by issued and/or pending patents.
RiboGuard™ RNase Inhibitor
T4 RNA Ligase
Terminator™ 5´-Phosphate-Dependent Exonuclease
Tobacco Acid Pyrophosphatase (TAP)
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