Study of Single-Stranded DNA Binding Protein-Nucleic Acids Interactions using Unmodified Gold Nanoparticles and Its Application for Detection of Single Nucleotide Polymorphisms

摘要

We have developed a label-free homogeneous phase bioassay to characterize the DNA binding properties of single-stranded DNA binding (SSB) protein, a key protein involved in various DNA processes such as DNA replication and repair. This assay uses gold nanoparticles (AuNPs) as sensing probe and is based on the phenomenon that preformed SSB-single-stranded DNA (ssDNA) complexes can protect AuNPs against salt-induced aggregation better than SSB or ssDNA alone. With the controlled particle aggregation/dispersion as measure, this assay can be used to detect the formation of SSB complexes with ssDNA of different length and nucleotide composition and to assess their binding properties without tedious and complicated assay procedures. On the basis of the inverse relationship between DNA hybridization efficiency and the tendency of SSB to form protection complex with unhybridized ssDNA to AuNPs, this assay is further developed to detect DNA hybridization with single nucleotide polymorphism selectivity. Owing to the high affinity between SSB and dissociated ssDNA, single-base mismatch discrimination in a long sequence of 30-mer DNA was achieved for both the end- and center-base mismatch. Unlike the conventional techniques for DNA and protein analysis, current AuNPs-based sensing strategy is simple in design, fast in detection, and economical for operation without the need of sophisticated equipment.