An Ultra-Stable and Dense Single-Molecule Click Platform for Sensing Protein-DNA Interactions

Abstract

AbstractWe demonstrate an ultra-stable, highly dense single-molecule assay ideal for observing protein-DNA interactions. Stable click Tethered Particle Motion (scTPM) leverages next generation click-chemistry to achieve an ultrahigh density of surface tethered reporter particles, has a high antifouling resistance, is stable at elevated temperatures to at least 45 °C, and is compatible with Mg2+, an important ionic component of many regulatory protein-DNA interactions. Prepared samples remain stable, with little degradation, for > 6 months in physiological buffers. These improvements enabled us to study previously inaccessible sequence and temperature dependent effects on DNA binding by the bacterial protein H-NS, a global transcriptional regulator found in E. Coli. This greatly improved assay can directly be translated to accelerate existing tethered particle based, single-molecule biosensing applications.