A DNA Force Circuit for Exploring Protein‐Protein Interactions at the Single‐Molecule Level^†

Abstract

Comprehensive SummaryProtein‐protein interactions (PPIs) play a crucial role in drug discovery and disease treatment. However, the development of effective drugs targeting PPIs remains challenging due to limited methodologies for probing their spatiotemporal anisotropy. Here, we propose a single‐molecule approach using a unique force circuit to investigate PPI dynamics and anisotropy under mechanical forces. Unlike conventional techniques, this approach enables the manipulation and real‐time monitoring of individual proteins at specific amino acids with defined geometry, offering insights into molecular mechanisms at the single‐molecule level. The DNA force circuit was constructed using click chemistry conjugation methods and genetic code expansion techniques, facilitating orthogonal conjugation between proteins and nucleic acids. The SET domain of the MLL1 protein and the tail of histone H3 were used as a model system to demonstrate the application of the DNA force circuit. With the use of atomic force microscopy and magnetic tweezers, optimized assembly procedures were developed. The DNA force circuit provides an exceptional platform for studying the anisotropy of PPIs and holds promise for advancing drug discovery research targeted at PPIs.