Single-molecule studies on DNA origami substrates

The DNA origami technique enables the rapid, high-yield assembly of arbitrarily shaped DNA nanostructures while providing an extraordinary degree of spatial and structural control. These nanostructures can be used as substrates for the precise arrangement of single molecules into well-defined assemblies which allows the visualization of chemical and biochemical reactions at a single-molecule level by atomic force microscopy. This proposal aims at further exploiting the great potential of the DNA origami technique and developing a versatile platform for the quantitative single-molecule study of medically relevant photochemical and protein-binding reactions in complex systems. The first part of the proposal focuses on the formation of cyclobutane pyrimidine dimers (CPDs) in DNA which represents the molecular origin of UVA-induced skin carcinogenesis. Initial studies have qualitatively shown that UVinduced CPD formation is affected by DNA topology whichleads to the characteristic periodicity of CPD appearance in nucleosomal DNA. Systematic investigations, however, are not available. Therefore, the influence of DNA topology on UVA-induced CPD formation will be quantitatively investigated in this project. To this end, DNA origami substrates will be decorated with TT-containing hairpin structures and double strands of different curvature, as well as with freely suspended DNA strands exhibiting different degrees of tension. The second part of the project aims at establishing a DNA origami-based strategy for the quantitative screening of small protein-binding molecules. Stateof- the-art drug discovery strategies increasingly employ DNA-encoded chemical libraries (DELs) in order to identify potent ligands against protein targets. As a fragment-based screening approach, self-assembled DELs allow the study of bidentate effects by binding of two different pharmacophores to the same protein. DNA origami substrates on the other hand can display multiple pharmacophores in arbitrary yet well-defined geometries and thus enable the quantification of synergetic effects in target protein binding in dependence of ligand distance and arrangement. Furthermore, this strategy provides a label-free binding assay which avoids negative effects resulting from protein modification and immobilization in DEL selection experiments.