Unparalleled approaches to directly visualize DNA-binding proteins and biomolecular condensates.

Abstract

The structure, regulation, and maintenance of DNA is critical to cellular function. Detailed molecular insights into these processes are essential for understanding their associated pathological conditions, establishing and validating biological models, and developing novel therapeutics. As a result, direct, real-time observations of individual proteins interacting with DNA are needed to obtain a complete picture and to validate current biological models. Technologies, such as the C-Trap, offer an exciting opportunity to meet these challenges by enabling researchers to observe DNA processes in real-time at the single-molecule level. Biomolecular condensates are also critical to cellular function. For instance, phase separation of proteins and RNA can cause formation of membrane-less organelles - e.g. stress granules, RNA-transport granules - that allow for efficient interactions between those biomolecules. There is increasing evidence that membrane-less condensates are implicated in human diseases such as cancer, amyotrophic lateral sclerosis, and Alzheimer's disease. Understanding the formation, physical properties, and mechano-chemical interactions of membrane-less organelles will provide essential information about their molecular basis and associated pathologies. Here, we present our efforts to enable unparalleled molecular-level discoveries using a combination of optical tweezers with fluorescence microscopy. We present examples in which our technologies enhanced the understanding of DNA regulators, DNA repair mechanisms, and DNA editing tools (e.g. CRISPR/Cas) in previously unattainable ways. We also show novel assays to investigate the properties and behavior of biomolecular condensates. Importantly, we show that advances in single-molecule technologies can be turned into easy-to-use instruments that enable discoveries previously reserved for the world's most specialized biophysics labs.