DNA origami as a tool for single-molecule fluorescence studies

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 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 future, light-based circuitry
could help tackling limitations of current electronics. Exploiting
the remarkable addressability of DNA origami objects, four
spectrally distinct fluorophores were incorporated into a
rectangular DNA origami at specific positions to create a
spectroscopic network. The unique feature of this arrangement is
that the energy transfer path can be manipulated by a mediator dye
that guides the light to two spectrally distinct outputs. To
visualize this control over the energy transfer path and for
sorting of the subpopulations, a new experimental four-color FRET
technique is developed, based on alternating laser excitation.