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Nanopore Analysis of Single-Stranded Binding Protein Interactions with DNA
Michael M Marshall1,2, Jan Ruzicka1,2, Osama K Zahid1,2
1†Joint School of Nanoscience and Nanoengineering and ‡Center for Biotechnology, Genomics, and Health Research, University of North Carolina at Greensboro, Greensboro, North Carolina 27401, United States.
Langmuir : the ACS Journal of Surfaces and Colloids
|April 4, 2015
Summary
We used nanopore technology to study how E. coli single-stranded binding protein (SSB) binds to DNA. Our findings show SSB can differentiate ssDNA from dsDNA, revealing insights into nucleoprotein filament formation and structure.
Area of Science:
- Biophysics
- Molecular Biology
- Nanotechnology
Background:
- Single-stranded DNA binding proteins (SSBs) are crucial for DNA replication, repair, and recombination in bacteria like E. coli.
- Understanding SSB-DNA interactions is fundamental to comprehending genome stability and cellular processes.
- Existing methods for studying these interactions can be limited in throughput or resolution.
Purpose of the Study:
- To investigate the binding kinetics and stoichiometry of E. coli SSB (EcoSSB) with single-stranded DNA (ssDNA) using a solid-state nanopore assay.
- To differentiate between free SSB, ssDNA, double-stranded DNA (dsDNA), and SSB-ssDNA complexes.
- To explore the structural characteristics of SSB-nucleoprotein filaments and binding cooperativity.
Main Methods:
- Utilized a solid-state nanopore platform to measure electrical translocation events of biomolecules.
- Analyzed changes in ionic current blockade amplitude and duration to characterize SSB-ssDNA interactions.
- Systematically varied SSB concentration relative to ssDNA to study filament formation and compared circular and linearized ssDNA.
Main Results:
- Successfully distinguished saturated SSB-nucleoprotein complexes from individual components (free SSB, ssDNA, dsDNA).
- Demonstrated high-fidelity differentiation of ssDNA from dsDNA in mixtures, leveraging SSB's high affinity for ssDNA.
- Observed a shift in electrical event amplitude correlating with increasing SSB concentration, indicative of weakly cooperative binding.
- Inferred structural details of the SSB-nucleoprotein complex by comparing binding to circular and linearized ssDNA.
Conclusions:
- Solid-state nanopore assays provide a sensitive and high-throughput method for studying protein-DNA interactions.
- EcoSSB exhibits high affinity and specificity for ssDNA, enabling robust differentiation from dsDNA.
- The study provides insights into the cooperative binding nature of SSB and the structural organization of nucleoprotein filaments.
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