Nanopore Analysis of Single-Stranded Binding Protein Interactions with DNA

We study the binding of E. coli single-stranded binding protein (SSB) to single-stranded DNA (ssDNA) using a solid-state nanopore assay. We find that saturated nucleoprotein complexes can be distinguished easily from free SSB, ssDNA, or double-stranded DNA individually and demonstrate that the high...

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Bibliographic Details
Published inLangmuir Vol. 31; no. 15; pp. 4582 - 4588
Main Authors Marshall, Michael M, Ruzicka, Jan, Zahid, Osama K, Henrich, Vincent C, Taylor, Ethan W, Hall, Adam R
Format Journal Article
LanguageEnglish
Published United States American Chemical Society 21.04.2015
Subjects
DNA, Single-Stranded - chemistry
DNA-Binding Proteins - chemistry
Electrochemical Techniques
Escherichia coli
Escherichia coli - chemistry
Escherichia coli Proteins - chemistry
Nanopores
Nucleoproteins - chemistry
Osmolar Concentration
Protein Binding
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Summary:We study the binding of E. coli single-stranded binding protein (SSB) to single-stranded DNA (ssDNA) using a solid-state nanopore assay. We find that saturated nucleoprotein complexes can be distinguished easily from free SSB, ssDNA, or double-stranded DNA individually and demonstrate that the high affinity of SSB for ssDNA can be exploited to achieve high-fidelity differentiation from duplex molecules in a mixture. We then study nucleoprotein filament formation by systematically varying the amount of SSB relative to ssDNA. We observe a concomitant shift in the mean amplitude of electrical events that is consistent with weakly cooperative binding. Finally, we compare circular and linearized ssDNA saturated with SSB and use the results to infer structural details of the nucleoprotein complex.
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ISSN:0743-7463
1520-5827
DOI:10.1021/acs.langmuir.5b00457