2008 NSTI Nanotechnology Conference and Trade Show - Nanotech 2008 - 11th Annual

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TechConnect Summit
Clean Technology 2008

Genetically engineered M13 bacteriophages as standalone tethers for probing single-molecule protein-DNA interactions

A.S. Khalil, J.Y. Mao, D. Ghosh, M.J. Lang, A.M. Belcher
Massachusetts Institute of Technology, US

Keywords:
single molecule, zinc finger, bacteriophage

Abstract:
Zinc fingers, the highly conserved, widespread, and multifunctional DNA binding domains for transcription factors, are paradigm proteins for studying protein-DNA interactions. Furthermore, as regulators of eukaryotic gene expression, the ability of these proteins to bind their complementary DNA substrate is crucial to cellular proliferation and development. Mutations in zinc finger proteins often lead to serious diseases, such as in the Cys2-His2 superfamily, which have been implicated in certain cancers, Huntington’s disease, and hereditary neuropathies. The biochemical features of this superfamily of proteins have been well documented and their crystal structures solved. However, biophysical investigations on the single-molecule level are lacking, perhaps due to an inability to build single-molecule assays around them. We report the genetic engineering of M13 filamentous bacteriophage into a standalone tether, specifically by expressing the DNA binding domain of Zif268/Egr1 directly on coat proteins of one end and biotin acceptor peptide (BAP) on coat proteins of the other end. We then probed the binding interaction between Zif268/Egr1 and its 9 base-pair GC-rich DNA target in a single-molecule optical trap setup, specifically by affixing short DNA strands on coverslip surfaces with a biotin-streptavidin linkage and allowing them to interact with zinc fingers, coupled to polystyrene beads via the M13 filament. This scheme allows us to build zinc finger binding specificity through high resolution measurements, with potential implications for gene therapy applications and a better understanding of transcription events involving low-level expression systems. Furthermore, our genetically encoded tether provides a testbed for mutation studies that could offer a glimpse of the single-molecule binding physics that underlie important disease states.


Nanotech 2008 Conference Program Abstract