SUMMARY Central functions in DNA biology and biotechnology are carried out by nucleoprotein machines. In these dynamic macromolecular assemblies, the DNA duplex is bound and distorted in complex with protein and sometimes RNA. Biophysical measurements and models are needed to understand the mechanisms of these machines, in which coordinated conformational changes in protein and nucleic acid components are coupled with chemical steps such as backbone cleavage or nucleotide hydrolysis. This is a renewal application for a grant in which we previously developed high-resolution and multimodal single-molecule approaches and applied them to elucidate mechanochemical coupling in the ATP-dependent supercoiling motor DNA gyrase from E. coli. Here, we propose to leverage our methods and insights to dissect the dynamics and mechanics of additional nucleoprotein machines, focusing on the RNA-guided nucleases Cas9 and Cas12a and comparing DNA gyrase motors across species. We will characterize substeps in DNA interrogation and DNA supercoiling, molecular determinants of energy landscapes and kinetics, and the effects of mechanical strains experienced in the genome. If successful, the project will determine the physical mechanisms of DNA interrogation by RNA-guided nucleases in dynamic and mechanical detail, providing a quantitative description of the target search process for enzymes that are currently being exploited for gene editing and for a rapidly expanding set of other applications involving specific targeting of activities to sites in the genome. New DNA gyrase measurements will further elucidate biophysical specializations, structural properties, and mechanical regulation of enzymes that are important targets for antibacterial drugs. Finally, single-molecule methods development driven by these biophysical questions will have broad applications in systems ranging from transcription to nucleosome remodeling.