Multi-parameter protein analysis with an electro-switchable biosurface

Auteurs

• Andreas Langer

Paramètres

• Manuels
• Manuels universitaires

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Measurements in stationary or mobile phases are essential for protein analysis. While immobilizing molecules on solid supports facilitates parallel interaction analysis, properties like size or shape are typically inferred from molecular mobility under external forces. These principles are often mutually exclusive, necessitating a multi-step workflow for comprehensive protein characterization. This dissertation demonstrates how to reconcile these measurement modalities by tethering proteins to surfaces using dynamically actuated nanolevers. Short DNA strands, activated by alternating electric fields, serve as capture probes for target proteins. By oscillating the proteins over nanometer amplitudes and comparing their motion to a theoretical model, the protein diameter can be accurately quantified. Structural alterations and conformational changes in proteins are readily detected. The versatility of electrically switchable DNA layers for high-throughput screening of low molecular weight compounds is illustrated through studies on human dihydrofolate reductase. An affinity ranking of small molecules is achieved via a competitive assay, revealing conformational changes in the enzyme complex. Additionally, the chip-based method is applied to study DNA polymerases and their interactions with nucleic acids. Real-time monitoring of DNA polymerase binding through changes in DNA motion allows for the determination of chemical rate cons

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• Année de publication: 2015