Ribonucleoprotein purification and characterization using RNA Mango

• DOI: 10.1261/rna.062166.117
• Publication Date: 2017-07-27T00:55:24Z
• Authors: Shanker Shyam S. ...; Matthew L. Ferguson; Eric J. Hayden; Xin Chen; Aaron A. Hoskins; Peter J. Unrau
• Keywords: Aptamer; Small nuclear RNA

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