Summary
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 catalyti-cally 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.
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Acknowledgments
We thank Christian Wenz for his contributions to the described protocols. This work was supported by an ISREC PhD fellowship to I.K., an EMBO Long-Term Postdoctoral Fellowship to G.C., and a Swiss National Science Foundation grant to J.L.
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© 2008 Humana Press, a part of Springer Science + Business Media, LLC
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Kurth, I., Cristofari, G., Lingner, J. (2008). An Affinity Oligonucleotide Displacement Strategy to Purify Ribonucleoprotein Complexes Applied to Human Telomerase. In: Lin, RJ. (eds) RNA-Protein Interaction Protocols. Methods in Molecular Biology, vol 488. Humana Press. https://doi.org/10.1007/978-1-60327-475-3_2
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DOI: https://doi.org/10.1007/978-1-60327-475-3_2
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