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Int. J. Mol. Sci. 2015, 16(9), 22456-22472; doi:10.3390/ijms160922456

Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1

Department of Microbiology, Faculty of Preclinical Medicine, the Fourth Military Medical University, Xi'an 710032, China
These authors contributed equally to this manuscript.
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Authors to whom correspondence should be addressed.
Academic Editor: Christo Z. Christov
Received: 22 June 2015 / Revised: 5 August 2015 / Accepted: 5 August 2015 / Published: 16 September 2015
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Abstract

Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs). The streptavidin-binding aptamer S1 sequence was inserted into the 3′ end of dengue virus (DENV) 5′–3′ UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. Several novel host DENV RBPs were subsequently identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which we further implicate in DENV replication. We proposed efficient S1 aptamer-based isolation of viral assembled RNPs from living mammalian cells will be generally applicable to the purification of high- and low-affinity RBPs and RNPs under endogenous conditions. View Full-Text
Keywords: living mammalian cells; DENV; streptavidin aptamer S1; affinity purification; RNA binding protein; 40S ribosomal protein S8 living mammalian cells; DENV; streptavidin aptamer S1; affinity purification; RNA binding protein; 40S ribosomal protein S8
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MDPI and ACS Style

Dong, Y.; Yang, J.; Ye, W.; Wang, Y.; Ye, C.; Weng, D.; Gao, H.; Zhang, F.; Xu, Z.; Lei, Y. Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1. Int. J. Mol. Sci. 2015, 16, 22456-22472.

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