We examine the effect of electronic coupling on the optical properties of Cy3 dimers attached to DNA duplexes as a function of base pair (bp) separation using steady-state and time-resolved spectroscopy. For close Cy3-Cy3 separations, 0 and 1 bp between dyes, intermediate to strong electronic coupling is revealed by modulation of the absorption and fluorescence properties including spectral band shape, peak wavelength, and excited-state lifetime. Using a vibronic exciton model, we estimate coupling strengths of 150 and 266 cm for the 1 and 0 bp separations, respectively, which are comparable to those found in natural light-harvesting complexes. For the strongest electronic coupling (0 bp separation), we observe that the absorption band shape is strongly affected by the base pairs that surround the dyes, where more strongly hydrogen-bonded G-C pairs produce a red-shifted absorption spectrum consistent with a J-type dimer. This effect is studied theoretically using molecular dynamics simulation, which predicts an in-line dye configuration that is consistent with the experimental J-type spectrum. When the Cy3 dimers are in a standard aqueous buffer, the presence of relatively strong electronic coupling is accompanied by decreased fluorescence lifetime, suggesting that it promotes nonradiative relaxation in cyanine dyes. However, we show that the use of a viscous solvent can suppress this nonradiative recombination and thereby restore the dimer fluorescent emission. Ultrafast transient absorption measurements of Cy3 dimers in both standard aqueous buffer and viscous glycerol buffer suggest that sufficiently strong electronic coupling increases the probability of excited-state relaxation through a dark state that is related to Cy3 torsional motion.

In order to fully realize the potential of DNA nanotechnology, it is crucial to overcome the lack of robust analytical techniques that continue to hinder the purification and characterization of DNA-based structures. In this Feature, we provide a snapshot of the current state of metrological techniques in DNA nanotechnology and look forward to emerging technologies that may offer new ways to probe and visualize these complex structures.

Molecular excitons, which propagate spatially via electronic energy transfer, are central to numerous applications including light harvesting, organic optoelectronics, and nanoscale computing; they may also benefit applications such as photothermal therapy and photoacoustic imaging through the local generation of heat via rapid excited-state quenching. Here we show how to tune between energy transfer and quenching for heterodimers of the same pair of cyanine dyes by altering their spatial configuration on a DNA template. We assemble “transverse” and “adjacent” heterodimers of Cy5 and Cy5.5 using DNA Holliday junctions. We find that the transverse heterodimers exhibit optical properties consistent with excitonically interacting dyes and fluorescence quenching, while the adjacent heterodimers exhibit optical properties consistent with nonexcitonically interacting dyes and disproportionately large Cy5.5 emission, suggestive of energy transfer between dyes. We use transient absorption spectroscopy to show that quenching in the transverse heterodimer occurs via rapid nonradiative decay to the ground state (∼31 ps) and that in the adjacent heterodimer rapid energy transfer from Cy5 to Cy5.5 (∼420 fs) is followed by Cy5.5 excited-state relaxation (∼700 ps). Accessing such drastically different photophysics, which may be tuned on demand for different target applications, highlights the utility of DNA as a template for dye aggregation.

DNA self‐assembly has proven to be a highly versatile tool for engineering complex and dynamic biocompatible nanostructures from the bottom up with a wide range of potential bioapplications currently being pursued. Primary among these is healthcare, with the goal of developing diagnostic, imaging, and drug delivery devices along with combinatorial theranostic devices. The path to understanding a role for DNA nanotechnology in biomedical sciences is being approached carefully and systematically, starting from analyzing the stability and immune‐stimulatory properties of DNA nanostructures in physiological conditions, to estimating their accessibility and application inside cellular and model animal systems. Much remains to be uncovered but the field continues to show promising results toward developing useful biomedical devices. This review discusses some aspects of DNA nanotechnology that makes it a favorable ingredient for creating nanoscale research and biomedical devices and looks at experiments undertaken to determine its stability in vivo. This is presented in conjugation with examples of state‐of‐the‐art developments in biomolecular sensing, imaging, and drug delivery. Finally, some of the major challenges that warrant the attention of the scientific community are highlighted, in order to advance the field into clinically relevant applications.