- Conspectus An important effort in the DNA nanotechnology field is focused on the rational design and manufacture of molecular structures and dynamic devices made of DNA. As is the case for other technologies that deal with manipulation of matter, rational development requires high quality and informative feedback on the building blocks and final products. For DNA nanotechnology such feedback is typically provided by gel electrophoresis, atomic force microscopy (AFM), and transmission electron microscopy (TEM). These analytical tools provide excellent structural information; however, usually they do not provide high-resolution dynamic information. For the development of DNA-made dynamic devices such as machines, motors, robots, and computers this constitutes a major problem. Bulk-fluorescence techniques are capable of providing dynamic information, but because only ensemble averaged information is obtained, the technique may not adequately describe the dynamics in the context of complex DNA devices. The single-molecule fluorescence (SMF) technique offers a unique combination of capabilities that make it an excellent tool for guiding the development of DNA-made devices. The technique has been increasingly used in DNA nanotechnology, especially for the analysis of structure, dynamics, integrity, and operation of DNA-made devices; however, its capabilities are not yet sufficiently familiar to the community. The purpose of this Account is to demonstrate how different SMF tools can be utilized for the development of DNA devices and for structural dynamic investigation of biomolecules in general and DNA molecules in particular. Single-molecule diffusion-based Förster resonance energy transfer and alternating laser excitation (sm-FRET/ALEX) and immobilization-based total internal reflection fluorescence (TIRF) techniques are briefly described and demonstrated. To illustrate the many applications of SMF to DNA nanotechnology, examples of SMF studies of DNA hairpins and Holliday junctions and of the interactions of DNA strands with DNA origami and origami-related devices such as a DNA bipedal motor are provided. These examples demonstrate how SMF can be utilized for measurement of distances and conformational distributions and equilibrium and nonequilibrium kinetics, to monitor structural integrity and operation of DNA devices, and for isolation and investigation of minor subpopulations including malfunctioning and nonreactive devices. Utilization of a flow-cell to achieve measurements of dynamics with increased time resolution and for convenient and efficient operation of DNA devices is discussed briefly. We conclude by summarizing the various benefits provided by SMF for the development of DNA nanotechnology and suggest that the method can significantly assist in the design and manufacture and evaluation of operation of DNA devices.