A method is described for immobilizing double-stranded DNAs to a planar gold surface with high density (1-3×1012 DNA/cm2, depending on their length) and uniform spacing (approx. 4 nm closest possible DNA-DNA separation). This is accomplished by adsorbing biotinylated DNAs onto a nearly close-packed monolayer of the protein streptavidin. This streptavidin monolayer, which offers approx. 5×1012 biotin sites per cm2, is prepared first by adsorbing it onto a mixed self-assembled monolayer on gold which contains biotin-terminated and oligo(ethylene glycol)-terminated alkylthiolates in a 3/7 ratio. This DNA-functionalized surface resists non-specific protein adsorption and is useful for probing the kinetics and equilibrium binding of proteins to DNA with surface plasmon resonance. This is demonstrated with the Mnt protein, which is found to bind in 3.8:1 ratio to its immobilized DNA operator sequence. This is consistent with its behavior in homogeneous solution, where it binds as a tetramer to its DNA. 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 double-stranded DNAs which are immobilized to gold with this streptavidin linker layer.
|Number of pages||9|
|Journal||Proceedings of SPIE - The International Society for Optical Engineering|
|State||Published - Jan 1 2000|
|Event||Scanning and Force Microscopies for Biomedical Applications II - San Jose, CA, USA|
Duration: Jan 23 2000 → Jan 24 2000