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 P_i

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´ + P_i

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 + 2P_i

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 (P_i) 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.