PROJECT SUMMARY/ABSTRACT We are requesting funds to purchase a LUMICKS C-Trap optical tweezers instrument, which is an integrated solution for single-molecule biochemistry and biophysics investigations that incorporates three-color fluorescence microscopy, optical tweezers, and five-channel microfluidics with a custom user interface for instrument control and automation. The capabilities of the C-Trap are broad, enabling the real-time observation and analysis of protein-protein and protein-nucleic acid interactions, liquid-liquid phase separation, protein folding and conformational changes, cell mechanics and response, and cellular structure and transport. The anticipated user base is equally broad, including chromosome biologists, molecular geneticists, structural virologists, and chemical biologists. Access to the C-Trap will quickly facilitate single-molecule experiments involving fundamental investigations into the mechanisms of DNA repair, transcriptional regulation, protein trafficking and quality control, DNA replication, horizontal gene transfer, the subcellular localization of biomolecules, and small-molecule targeting of proteins. In the long term, the data gathered using the C-Trap will help a large group of NIH-supported researchers to make progress in understanding basic cancer biology, host- pathogen interactions, antibiotic resistance, protein (un)folding diseases, and an array of developmental and neurological pathologies related to the protein families being studied. We further anticipate that the C-Trap can be used to foster a community of single-molecule researchers at Indiana University and neighboring institutions, as well as to train the next generation of single-molecule experimentalists. Overall, the purchase of the C-Trap will lead to high-impact publication in top tier journals, enabling users to better compete for extramural funding and make a positive impact in their fields of research and human health.