A: RNA 5′ Polyphosphatase is an enzyme that removes the γ and β phosphate groups from 5′-triphosphorylated or 5′-diphosphorylated RNA, converting these molecules into 5′-monophosphorylated RNA. Capped RNA is not a substrate for this enzyme.
A: Yes. When used in combination with XRN1, RNA 5′ Polyphosphatase converts uncapped RNA into 5′-monophosphorylated RNA, which is subsequently degraded by XRN1. Capped mRNA remains intact, enabling enrichment of high-quality capped transcripts.
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A: Inorganic phosphate, a natural reaction product, inhibits enzyme activity. Excess free nucleotides may also reduce effective activity by acting as competing substrates. Thorough RNA cleanup is recommended prior to treatment.
A: RNA should be purified to remove unincorporated nucleotides and inorganic phosphate from transcription reactions. Purified RNA should be resuspended in RNase-free water or RNase-free TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA).
A: Stable secondary structures near the 5′ end, such as hairpins or intermolecular duplexes, can limit enzyme access. Heat denaturation of RNA prior to the reaction or longer incubation times may improve conversion efficiency.
A: One unit is defined as the amount of enzyme required to release 1 nanomole of inorganic phosphate from ATP in 1 hour at 37°C under defined assay conditions.
A: No, 5’ RNA Polyphosphatase is Mg2+ independent and therefore does not require additional Mg2+ for activity.
A: RNA 5′ Polyphosphatase is widely used in workflows involving RNA circularization, end-labeling, adapter ligation, RNA stability studies, and purification of capped mRNA for research, therapeutic development, and vaccine applications.
