Single-molecule fluorescence studies have become a routine practice in laboratories worldwide. As an experimental tool, especially fluorescence resonance energy transfer (FRET) has helped to unravel conformational changes and interactions of biomolecules. With the DNA origami method a new technique to create nanoscale shapes with DNA as a building material was recently introduced. As shown in this work, DNA nanotechnology can be readily combined with single-molecule FRET experiments, opening up

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... new scientific prospects. With the progress of single-molecule techniques, the limiting factor for many applications is the quality of individual dye molecules. For successful single-molecule experiments, an understanding of the photophysical properties of dyes is essential. The first part of this thesis is devoted to providing fundamental insights into characteristic properties of fluorescent molecules. The common feature of single-molecule blinking is studied for the homologous series of cyanine dyes. A model is presented that allows predicting the blinking behavior of fluorophores, based on parameters such as the redox potential and chromophore size. The predictions are experimentally verified by evaluating fluorescence time transients of immobilized dye molecules. To characterize the distance dependence of FRET, in the past several approaches have been made to build a molecular ruler, including double stranded DNA and the polypeptide polyproline as spacer molecules. It is demonstrated that the DNA origami technique allows creating tailored molecular spacers that are specifically engineered to meet experimental requirements. A rigid DNA origami block was designed that can be used as a reliable FRET ruler on the single-molecule level. This approach offers distinct advantages compared to previous systems that suffered from limited persistence lengths and sample heterogeneity. The final project in this thesis was guided by the vision to use a DNA origami structure as a breadboard for molecular photonic circuits. In the fut [...]