An Affinity Oligonucleotide Displacement Strategy to Purify Ribonucleoprotein Complexes Applied to Human Telomerase

• DOI: 10.1007/978-1-60327-475-3_2
• Publication Date: 2008-11-03T17:23:32Z
• Authors: Isabel Kurth; Gaël Cristofari; Joachim Lingner
• Keywords: 0301 basic medicine; 03 medical and health sciences; Ribonucleoproteins; Oligonucleotides; Humans; Models, Theoretical; Telomerase; Protocols; Chromatography, Affinity; Protein Binding

Antisense oligonucleotides have been used to study the structure and function of small nuclear ribonucleoprotein (snRNP) complexes and were adapted and modified for the purification of a variety of RNPs. We describe methods for recombinant expression and reconstitution of catalytically active human telomerase and the purification of native and recombinant telomerase using antisense affinity oligonucleotides. The purification procedure involves binding of the RNP complex to NeutrAvidin beads via a biotinylated 2'-O-methyl (2'-OMe) RNA oligonucleotide complementary to the RNA subunit. The complex is eluted from the beads through competition with a displacement oligonucleotide. Thus, the purified RNP is eluted under mild conditions, retaining its catalytic activity.