Interrogating Oestrogen Receptor–DNA Interactions using Metallic Nanoparticles and Surface Plasmon Resonance Technique

Abstract

Surface plasmon resonance (SPR) spectroscopy is an important technique for real-time measurement of biomolecular interactions on the solid–liquid interface. Metallic nanoparticles (mNP) have unique optical properties arising from localized SPR. They can be used as elegant colorimetric probes for studying biomolecular interactions in homogenous solution. In this study, we used the two techniques in parallel to study the binding interactions of oestrogen receptors (ERα and ERβ) with their response elements (ERE). First, we have validated the principle of a gold nanoparticle (AuNP)-based colorimetric assay that protein–DNA complexes are able to provide more electrosteric forces than protein alone, using AuNP of a range of particle size (10, 20, and 50 nm) and examined particle size-dependent assay performance for detecting ERβ­­–DNA interactions. Second, we have demonstrated the ability of this AuNP assay to discriminate subtle binding affinity difference of ERβ to a mutated ERE containing a single base pair alternation relative to that with a perfect ERE sequence. The accuracy and sensitivity of the AuNP assay for detecting sequence dependent binding are found comparable with the golden standard SPR assay. Third, through a parallel study, we have compared the performance of the AuNP assay and SPR assay for measuring a protein–DNA binding event when the protein is partially denatured or has lower reactivity. We found that the AuNP assay can detect protein–DNA complex formation and determine sequence specificity more easily owing presumably to the homogenouse phase binding nature. With this study we conclude that the AuNP-based assay is a valuable complementary technique to SPR for interrogating sequence-specific protein–DNA interactions.