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.