Evaluation of two- and three-dimensional streptavidin binding platforms for surface plasmon resonance spectroscopy studies of DNA hybridization and protein-DNA binding.
Surface plasmon resonance (SPR) spectroscopy has been used to study DNA assembly, DNA hybridization, and protein-DNA interactions on two streptavidin (SA) sensor chips. On one chip, SA molecules are immobilized on a biotin-exposed surface, forming an ordered two-dimensional (2D) SA monolayer. The other chip, BIAcore's SA chip, contains SA molecules immobilized within a three-dimensional (3D) carboxylated dextran matrix. Compared to the 2D chip, the 3D SA matrix allows for a slower immobilization rate of biotinylated DNA due to diffusion limitation in the dextran matrix, but with twice the amount of the immobilized DNA due to the greater number of reactive sites, which in turn enables a higher sensitivity for DNA hybridization detection. Interestingly, having a greater DNA probe dispersion in the 3D matrix does not induce a higher DNA hybridization efficiency. In a study of protein binding to immobilized DNA (estrogen receptor to estrogen response elements), aiming at assessing the DNA sequence dependent protein binding behavior, the 2D and 3D chips produce different binding characteristics. On the 2D chip, the protein binding exhibits a better selectivity to the specific sequences, regardless of binding stringency (e.g. salt concentration), whereas on the 3D chip, the liquid handling system needs to be optimized in order to minimize transport limitations and to detect small affinity differences. Through this study we demonstrate that the physicochemical structure of SPR chips affects the apparent binding behaviors of biomolecules. When interpreting SPR binding curves and selecting a sensor chip, these effects should be taken into account.
Yang
Nan
N
Department of Materials Science and Engineering, Massachusetts Institute of Technology, United States.
Su
Xiaodi
X
Tjong
Vinalia
V
Knoll
Wolfgang
W
D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D017403:In Situ Hybridization / Q000295:instrumentation / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods*
D004268:DNA-Binding Proteins; D004247:DNA