DNA skybridge: 3D structure producing a light sheet for high-throughput single-molecule imaging.

Real-time visualization of single-proteins or -complexes on nucleic acid substrates is an essential tool for characterizing nucleic acid binding proteins. Here, we present a novel surface-condition independent and high-throughput single-molecule optical imaging platform called 'DNA skybridge'. The DNA skybridge is constructed in a 3D structure with 4 µm-high thin quartz barriers in a quartz slide. Each DNA end is attached to the top of the adjacent barrier, resulting in the extension and immobilization of DNA. In this 3D structure, the bottom surface is out-of-focus when the target molecules on the DNA are imaged. Moreover, the DNA skybridge itself creates a thin Gaussian light sheet beam parallel to the immobilized DNA. This dual property allows for imaging a single probe-tagged molecule moving on DNA while effectively suppressing interference with the surface and background signals from the surface.

Discovering anomalous hybridization kinetics on DNA nanostructures using single-molecule fluorescence microscopy.

DNA nanostructures are finding diverse applications as scaffolds for molecular organization. In general, components such as nucleic acids, proteins, and nanoparticles are attached to addressable DNA nanostructures via hybridization, and there is interest in exploiting hybridization for localized computation on DNA nanostructures. This report details two fluorescence microscopy methods, single-particle fluorescence resonance energy transfer (spFRET) and DNA-PAINT (points accumulation for imaging in nanoscale topography), that have been successfully used to detect anomalies of hybridization reactions on individual DNA nanostructures. We compare and contrast the two techniques, highlighting their respective strengths in studying equilibrium and non-equilibrium hybridization as well as assessing the variability of behaviors within individual nanostructures and across a population of nanostructures.

Quantification of cellular uptake of DNA nanostructures by qPCR.

DNA nanostructures facilitating drug delivery are likely soon to be realized. In the past few decades programmed self-assembly of DNA building blocks have successfully been employed to construct sophisticated nanoscale objects. By conjugating functionalities to DNA, other molecules such as peptides, proteins and polymers can be precisely positioned on DNA nanostructures. This exceptional ability to produce modular nanoscale devices with tunable and controlled behavior has initiated an interest in employing DNA nanostructures for drug delivery. However, to obtain this the relationship between cellular interactions and structural and functional features of the DNA delivery device must be thoroughly investigated. Here, we present a rapid and robust method for the precise quantification of the component materials of DNA origami structures capable of entering cells in vitro. The quantification is performed by quantitative polymerase chain reaction, allowing a linear dynamic range of detection of five orders of magnitude. We demonstrate the use of this method for high-throughput screening, which could prove efficient to identify key features of DNA nanostructures enabling cell penetration. The method described here is suitable for quantification of in vitro uptake studies but should easily be extended to quantify DNA nanostructures in blood or tissue samples.