Poly(A) Polymerase

• Catalyzes the addition of AMP from ATP to the 3’ hydroxyl of RNA

Poly(A) Polymerase attaches ATP (catalyzed from AMP) to the 3ʹ hydroxyl of RNA, which is useful for making poly(A)-tailed RNA.

The enzyme is supplied in 25 mM Tris-HCl, 500 mM NaCl, 1 mM MgCl₂, 0.1 mM DTT, 0.1 mM EDTA and 50% glycerol; pH 8.0 at 25°C.

10X Poly(A) Polymerase Reaction Buffer (B7460) contains 500 mM Tris-HCl, 2.5 M NaCl and 100 mM MgCl₂; pH 7.9 at 25°C.

Poly(A) Polymerase catalyzes the addition of AMP from ATP to the 3ʹ hydroxyl of RNA. The reaction requires Mg²⁺ and is template independent.

Polymerase properties

• Storage temperature: –25°C to –15°C

Source of recombinant enzyme protein
The protein is produced by anE. coli strain expressing the Poly(A) Polymerase gene from a plasmid.

Unit definition: One unit is defined as the amount of enzyme that will incorporate 1 nmol of ATP into acid-insoluble material in 10 minutes at 37°C.

Molecular weight: 53,870 Daltons

Quality control analysis

Specific activity was measured using a twofold serial dilution method. Dilutions of enzyme were made in 1X reaction buffer (50 mM Tris-HCl, 250 mM NaCl, 10 mM MgCl₂, pH 7.9 at 25°C, and added to 50 µl reactions containing a 15-mer RNA oligo, 1X reaction buffer, 1 mM ATP, 2.5 mM MnCl₂ and 3H-ATP. Reactions were incubated 10 minutes at 37°C, place on ice, and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26).

Protein concentration is determined by OD₂₈₀ absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.

Single-stranded exonuclease is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease activity is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.

E. coli contamination is evaluated using 5 µl replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminatingE. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

Non-specific RNAse contamination is assessed using the RNAse Alert kit, (Integrated DNA Technologies), following the manufacturer’s guidelines.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• Making polyA tailed RNA
• 3’ end RNA labeling
• mRNA therapeutics