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Dephosphorylates 5´-triphosphorylated RNA, such as primary RNA transcripts, but does not desphosphorylate 5´-capped mRNA
- Specific: Removes the γ and β phosphates from 5´-triphosphorylated and the β phosphates from diphosphorylated RNA, but will not dephosphorylate monophosphorylated or 5´-capped RNA
- Heat Inactivated: Heat at 65°C for 20 minutes to kill enzyme activity
- Flexible: Use for various applications such as 5´-RNA ligation-tagging using T4 RNA Ligase, analysis of 5´-end structure of RNA, and preparation of substrate RNAs for subsequent degradation with 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´ to ; 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´ to ; 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 to ; (d)NMP + 2Pi
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 the Epicentre Terminator™ 5´-Phosphate-Dependent Exonuclease.
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.
