Surface plasmon resonance imaging studies of protein-carbohydrate interactions, protein-DNA interactions, and chemically induced hairpin formation in DNA monolayers

The application of surface plasmon resonance (SPR) imaging to study the interactions of biomolecules with carbohydrate and DNA microarrays is presented. SPR imaging is a label-free high-throughput detection method that is used to study biomolecular interactions that occur between probe molecules immobilized onto thin gold films and target biomolecules that adsorb to the film. A novel immobilization method in which thiol-modified probe molecules are attached onto gold films through the formation of a disulfide bond was used to generate DNA and carbohydrate microarrays suitable for SPR imaging studies of oligonucleotides and protein adsorption. Specifically, carbohydrate arrays fabricated with polydimethylsiloxane microchannels were used to study carbohydrate-protein interactions. SPR imaging measurements were employed to: (i) construct adsorption isotherms for the interactions of the lectins ConA and jacalin to the carbohydrate surfaces, (ii) monitor protein binding to surfaces presenting different compositions of the immobilized carbohydrates, and (iii) measure the solution equilibrium dissociation constants for ConA and jacalin towards mannose and galactose, respectively.; In addition, a previously reported maleimide attachment scheme and UV photopatterning were used to fabricate DNA arrays for the study of DNA-protein interactions and hairpin formation in DNA monolayers. SPR imaging was employed to study the sequence specific adsorption of OmpR and VanR bacterial response regulator proteins onto DNA arrays, and to: (i) monitor and compare the binding of both response regulators to various promoter regions on the DNA array, (ii) compare the binding of the OmpR response regulator protein in its phosphorylated and non-phosphorylated forms, and (iii) monitor the inhibition of VanR binding to the DNA arrays in the presence of a small molecule DNA binding inhibitor. Finally, the application of SPR imaging to study the intercalation of a naphthyridine dimer that stabilizes G-G mismatches in double stranded DNA is presented. The binding of the naphthyridine dimer to DNA microarrays was used to drive the formation of a hairpin structure in a DNA sequence specifically designed for this purpose.