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A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies
Published on: July 24, 2017
A bacterial one-hybrid selection system for interrogating zinc finger-DNA interactions
Sundar Durai1, Allen Bosley, Alice Bridgeman Abulencia
1Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
Combinatorial Chemistry & High Throughput Screening
|May 27, 2006
Summary
We developed bacterial one-hybrid systems to select zinc finger proteins that bind specific DNA sequences. These systems enable efficient screening and quantitative evaluation of zinc finger-DNA interactions.
Area of Science:
- Molecular Biology
- Biotechnology
- Genetics
Background:
- Zinc finger proteins (ZFPs) are crucial for regulating gene expression.
- Developing efficient methods to select ZFPs with specific DNA-binding affinities is essential for synthetic biology and gene therapy applications.
Purpose of the Study:
- To create and validate two novel bacterial one-hybrid systems for the selection and interrogation of zinc finger-DNA interactions.
- To enable the isolation of ZFPs targeting specific DNA motifs with high affinity and specificity.
Main Methods:
- Development of two bacterial one-hybrid systems using distinct reporter genes: chloramphenicol acetyltransferase (CAT) and green fluorescent protein (GFP).
- Construction of plasmids encoding a zinc finger fused to a RNA polymerase alpha subunit fragment and a reporter gene under the control of a ZFP-binding site.
- Utilizing a library of approximately 2 x 10^5 ZFP variants to select for binding to the 5'-GGGGCAGAA-3' motif.
Main Results:
- The CAT reporter system demonstrated a 10-fold increase in chloramphenicol resistance, facilitating single-step selection of sequence-specific ZFPs.
- The GFP reporter system showed an 8- to 22-fold increase in cell fluorescence, enabling quantitative analysis via flow cytometry and potential for positive/negative selection.
- Both systems successfully selected ZFPs with affinity for the target motif, reporting dissociation constants below approximately 1 microM.
Conclusions:
- The developed bacterial one-hybrid systems are effective tools for selecting and characterizing zinc finger proteins with specific DNA-binding capabilities.
- These systems offer versatile approaches for ZFP discovery, with applications in molecular biology research and the development of gene-targeting technologies.
