Pitt Scientists Develop New Method for Single-Molecule DNA-Binding Protein Analysis

A novel method for characterizing protein-DNA dynamics at the single-molecule level is reported in Nucleic Acids Research this week. Watching DNA-binding proteins interact with DNA substrates in real-time at the single-molecule level can yield valuable insights into how proteins detect and bind their targets. Aiming to build upon current approaches that use imaging techniques or optical platforms to resolve fluorescent proteins to the single-molecule level, a team from the University of Pittsburgh developed a technique dubbed SMADNE — short for single-molecule analysis of DNA-binding proteins from nuclear extracts — that allows DNA-binding proteins to be taken from a cell's nucleus and then analyzed using C-trap optical tweezers, microfluidics, and confocal microscopy. The scientists demonstrate SMADNE on undamaged DNA and three forms of DNA damage using seven native DNA repair proteins and two structural variants but write that the method can be extended to other types of DNA-binding proteins such as transcription factors and DNA polymerases. "Furthermore, this new approach could be used to observe macromolecular interactions from extracts generated from a wide range of cells and tissues from animals expressing fluorescent proteins," they add. "The SMADNE technique represents a novel, scalable, and universal method to obtain single-molecule mechanistic insights into key protein-DNA interactions in an environment containing physiologically-relevant nuclear proteins."