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Nucleic Acids Research Advance Access originally published online on September 12, 2008
Nucleic Acids Research 2008 36(19):e128; doi:10.1093/nar/gkn559
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Nucleic Acids Research, 2008, Vol. 36, No. 19 e128
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Cell-free selection of RNA-binding proteins using in vitro compartmentalization

Yu Chen1,*, Jana Mandic2 and Gabriele Varani1,2

1Department of Chemistry and 2Department of Biochemistry, University of Washington, Seattle WA, USA

*To whom correspondence should be addressed. Tel: +1 206 543 7113; Fax: +1 206 685 8665; Email: chenyu{at}u.washington.edu

Correspondence may also be addressed to G. Varani. Tel: +1 206 543 7113; Fax: +1 206 685 8665; Email: varani{at}chem.washington.edu

Received July 3, 2008. Revised August 11, 2008. Accepted August 18, 2008.

RNA-binding proteins (RBPs) perform many essential functions in the post-transcriptional control of gene expression. If we were able to engineer RBPs with new specificity, it would also become possible to develop new tools to control and investigate gene expression pathways. Molecular evolution methods such as phage display have been introduced to achieve this goal, but the large interface between these proteins and RNA relative to the size of library that can be constructed limits the efficacy of this method. In order to increase the diversity of libraries used for selection of RBPs, we applied the emulsion-based in vitro compartmentalization (IVC) method to select RBPs with defined specificity. A new approach was developed to link genotype and phenotype by fusing the target RBP to zinc finger proteins (ZFPs) that bind to a cognate DNA sequence inserted upstream of the promoter. The expressed fusion protein (ZFP–RBP) binds to its encoding DNA with high affinity via the ZFP target-binding site. After breaking the emulsion, the RBP can be selected based on its affinity for a biotinylated RNA bait. We demonstrate the effectiveness of this method that should enable the selection of RBPs with new specificity or improved affinity.


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