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PMID: 18790803 Published · ppublish English Evaluation Study Journal Article Research Support, N.I.H., Extramural

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

Nucleic acids research ·Vol. 36 ·No. 19 ·2008-11-00 ·Pages e128

Chen Y, Mandic J, Varani G

Abstract

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.

MeSH Terms
Directed Molecular Evolution/methods Gene Library Genotype Models, Molecular Mutation Phenotype Protein Engineering/methods Protein Structure, Tertiary RNA/chemistry RNA-Binding Proteins/chemistry,genetics Recombinant Fusion Proteins/chemistry Ribonucleoprotein, U1 Small Nuclear/chemistry,genetics Zinc Fingers
Chemicals
RNA-Binding Proteins Recombinant Fusion Proteins Ribonucleoprotein, U1 Small Nuclear U1A protein RNA
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Chen Yu
Department of Chemistry and Department of Biochemistry, University of Washington, Seattle WA, USA.
Mandic Jana
Varani Gabriele
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/gkn559
Published
2008-11-00
Epub
2008-00-12
Pages
e128
Language
English
Region
England
NLM ID
0411011
PMCID
PMC2577342
Subset
IM
Grants
NIGMS NIH HHS · R01 GM064440 · United States
External Links
PubMed source DOI: 10.1093/nar/gkn559 PMC Full Text View journal details
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