A Single Molecule Perspective on Protein-DNA Condensates

Biomolecular condensates are dynamic intracellular structural units or distinct reaction spaces that can form by condensation of their constituents from the cytoplasm or the nucleoplasm. It is generally not clear yet, how dynamic, continuum-like condensate properties relevant for large-scale intracellular organisation emerge from the interplay of proteins and nucleic acids on the level of few individual molecules. With this work, we expand the portfolio of methods to investigate the role of protein-nucleic acid interactions in biomolecular condensates by introducing optical tweezers-based mechanical micromanipulation of single DNA molecules combined with confocal fluorescence microscopy to the field. We used this approach to characterise how the two landmark proteins1 Fused in Sarcoma and Heterochromatin Protein 1 form condensates with single DNA molecules. Fused in Sarcoma (FUS) is a key protein for various aspects of the nucleic acid metabolism and evidence is accumulating that biomolecular condensation is crucial for both, its physiological functions and its role in pathological aggregate formation. In this thesis, we directly visualised the formation of FUS condensates with single molecules of ssDNA and dsDNA. We showed that the formation of these microcondensates is based on nucleic acid scaffolding. We explored their mechanical properties and found that the mechanical tension that (FUS dsDNA) condensates can withstand or exert is in the range below 2 pN. We further demonstrated that already on this fundamental scale and with limited amounts of constituent molecules, dynamic properties like shape relaxations, reminiscent of viscoelastic materials, can emerge. Heterochromatin Protein 1 (HP1) is a prototypic chromatin organising factor that is in particular involved in the formation of dynamically compacted heterochromatin domains. HP1 forms biomolecular condensates and compacts DNA strands in vitro. In this work, we measured the influence of HP1 on the mechanical properties of individual DNA molecules and demonstrated the response of HP1-DNA condensates to different environmental conditions. We contributed a methodological framework to characterise viscoelastic-like systems on the single molecule level. Taken together, our optical tweezers-based approach revealed structural and mechanical properties of prototypic protein-DNA condensates and hence helped to elucidate mechanisms underlying their formation in unprecedented spatiotemporal and mechanical detail. We anticipate that this method can become a valuable tool to investigate how large-scale intracellular organisation based on protein-nucleic acid condensation emerges from interactions between individual building blocks