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RNA. 2017 Oct;23(10):1592-1599. doi: 10.1261/rna.062166.117. Epub 2017 Jul 26.

Ribonucleoprotein purification and characterization using RNA Mango.

Author information

1
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
2
Department of Physics, Boise State University, Boise, Idaho 83725, USA.
3
Department of Biological Science and Biomolecular Sciences Graduate Program, Boise State University, Boise, Idaho 83725, USA.
4
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.

Abstract

The characterization of RNA-protein complexes (RNPs) is a difficult but increasingly important problem in modern biology. By combining the compact RNA Mango aptamer with a fluorogenic thiazole orange desthiobiotin (TO1-Dtb or TO3-Dtb) ligand, we have created an RNA tagging system that simplifies the purification and subsequent characterization of endogenous RNPs. Mango-tagged RNP complexes can be immobilized on a streptavidin solid support and recovered in their native state by the addition of free biotin. Furthermore, Mango-based RNP purification can be adapted to different scales of RNP isolation ranging from pull-down assays to the isolation of large amounts of biochemically defined cellular RNPs. We have incorporated the Mango aptamer into the S. cerevisiae U1 small nuclear RNA (snRNA), shown that the Mango-snRNA is functional in cells, and used the aptamer to pull down a U1 snRNA-associated protein. To demonstrate large-scale isolation of RNPs, we purified and characterized bacterial RNA polymerase holoenzyme (HE) in complex with a Mango-containing 6S RNA. We were able to use the combination of a red-shifted TO3-Dtb ligand and eGFP-tagged HE to follow the binding and release of the 6S RNA by two-color native gel analysis as well as by single-molecule fluorescence cross-correlation spectroscopy. Together these experiments demonstrate how the Mango aptamer in conjunction with simple derivatives of its flurophore ligands enables the purification and characterization of endogenous cellular RNPs in vitro.

KEYWORDS:

Mango; RNA; RNP pull-down; TO1; TO3; desthiobiotin; fluorophore; purification

PMID:
28747322
PMCID:
PMC5602116
DOI:
10.1261/rna.062166.117
[Indexed for MEDLINE]
Free PMC Article

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