AVS 47th International Symposium
    Biomaterial Interfaces Friday Sessions
       Session BI-FrM

Paper BI-FrM6
Probing Protein Interactions with Surface-Immobilized Double-Stranded DNA Using Surface Plasmon Resonance Sensing Techniques

Friday, October 6, 2000, 10:00 am, Room 202

Session: Biomolecular Recognition at Surfaces
Presenter: J.S. Shumaker-Parry, University of Washington
Authors: J.S. Shumaker-Parry, University of Washington
C.T. Campbell, University of Washington
K.E. Nelson, University of Washington
G.D. Stormo, Washington University Medical School
F.S. Silbaq, University of Colorado
R.H. Aebersold, University of Washington
Correspondent: Click to Email
Understanding the molecular mechanisms of gene expression in eukaryotes requires a precise knowledge of the strength and specificity of protein:DNA interactions. Surface plasmon resonance sensing techniques are important for monitoring the adsorption of biomolecules from liquid solutions onto functionalized solid surfaces with high sensitivity and fast time response. Simple methods convert changes in the angle or wavelength at which the surface plasmon resonance (SPR) of a thin metal film is optically excited into adsorbate concentrations. Methods for monitoring interactions between the transcription repressor protein Mnt and surface-immobilized double-stranded DNA using SPR spectroscopy and microscopy will be described. We have immobilized dsDNAs onto a planar gold surface with high density (1-3x10@super 12@ DNA/cm@super 2@, depending on their length) and uniform spacing (~4 nm closest possible DNA-DNA separation). This was accomplished by adsorbing biotinylated DNAs onto a nearly close-packed monolayer of the protein streptavidin prepared first by adsorbing it onto a mixed self-assembled monolayer on gold containing biotin-terminated and oligo(ethylene glycol)-terminated alkylthiolates in a 3/7 ratio. This DNA-functionalized surface resists non-specific protein adsorption. SPR spectroscopy experiments have shown that Mnt binds in 3.8:1 ratio to its immobilized DNA recognition sequence. This is consistent with its behavior in homogeneous solution, where it binds as a tetramer to its DNA binding sequence. A sequence with a single base-pair mutation shows nearly as much Mnt binding, but a completely random DNA sequence shows only 5% of this binding. This proves that DNA-binding proteins bind sequence-specifically to dsDNAs that are immobilized to gold with this streptavidin linker layer. SPR microscopy is being developed to extend these studies to probe protein interactions with an array of dsDNA-containing elements immobilized on a sensor surface.