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Abstract The label-free identification of individual proteins from liquid samples by surface-enhanced Raman scattering (SERS) spectroscopy is a highly desirable goal in biomedical diagnostics. However, the small Raman scattering cross-section of most (bio-)molecules requires a means to strongly amplify their Raman signal for successful measurement, especially for single molecules. This amplification can be achieved in a plasmonic hotspot that forms between two adjacent gold nanospheres. However, the small (≈1−2 nm) gaps typically required for single-molecule measurements are not accessible for most proteins. A useful strategy would thus involve dimer structures with gaps large enough to accommodate single proteins, whilst providing sufficient field enhancement for single-molecule SERS. Here, we report on using a DNA origami scaffold for tip-to-tip alignment of gold nanorods with an average gap size of 8 nm. The gaps are accessible to streptavidin and thrombin, which are captured at the plasmonic hotspot by specific anchoring sites on the origami template. The field enhancement achieved for the nanorod dimers is sufficient for single-protein SERS spectroscopy with sub-second integration times. This design for SERS probes composed of DNA origami with accessible hotspots promotes future use for single-molecule biodiagnostics in the near-infrared range.

Abstract Liquid droplets of biomolecules play key roles in organizing cellular behavior, and are also technologically relevant, yet physical studies of dynamic processes of such droplets have generally been lacking. Here, we investigate and quantify the dynamics of formation of dilute internal inclusions, i.e., vacuoles, within a model system consisting of liquid droplets of DNA ‘nanostar’ particles. When acted upon by DNA-cleaving restriction enzymes, these DNA droplets exhibit cycles of appearance, growth, and bursting of internal vacuoles. Analysis of vacuole growth shows their radius increases linearly in time. Further, vacuoles pop upon reaching the droplet interface, leading to droplet motion driven by the osmotic pressure of restriction fragments captured in the vacuole. We develop a model that accounts for the linear nature of vacuole growth, and the pressures associated with motility, by describing the dynamics of diffusing restriction fragments. The results illustrate the complex non-equilibrium dynamics possible in biomolecular condensates.

Here, the authors experimentally demonstrate chiral transfer over large distances up to 100 nm. They realise the coupling with an achiral nanosphere situated between a pair of distant gold nanorods arranged in a chiral fashion using DNA origami, and observe enhanced circular dichroism signals.

TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor are important constituents of all eukaryotic initiation complexes. Here, the authors use a DNA origami-based force clamp to investigate the assembly dynamics of human initiation complexes in the RNAP II and RNAP III systems at the single-molecule level under pico newton forces.

Since the arrival of DNA nanotechnology nearly 40 years ago, the field has progressed from its beginnings of envisioning rather simple DNA structures having a branched, multi-strand architecture into creating beautifully complex structures comprising hundreds or even thousands of unique strands, with the possibility to exactly control the positions down to the molecular level. While the earliest construction methodologies, such as simple Holliday junctions or tiles, could reasonably be designed on pen and paper in a short amount of time, the advent of complex techniques, such as DNA origami or DNA bricks, require software to reduce the time required and propensity for human error within the design process. Where available, readily accessible design software catalyzes our ability to bring techniques to researchers in diverse fields and it has helped to speed the penetration of methods, such as DNA origami, into a wide range of applications from biomedicine to photonics. Here, we review the historical and current state of CAD software to enable a variety of methods that are fundamental to using structural DNA technology. Beginning with the first tools for predicting sequence-based secondary structure of nucleotides, we trace the development and significance of different software packages to the current state-of-the-art, with a particular focus on programs that are open source.

Rod-like and sheet-like nano-particles made of desoxyribonucleic acid (DNA) fabricated by the DNA origami method (base sequence-controlled self-organized folding of DNA) are dispersed in a lyotropic chromonic liquid crystal made of an aqueous solution of disodium cromoglycate. The respective liquid crystalline nanodispersions are doped with a dichroic fluorescent dye and their orientational order parameter is studied by means of polarized fluorescence spectroscopy. The presence of the nano-particles is found to slightly reduce the orientational order parameter of the nematic mesophase. Nano-rods with a large length/width ratio tend to preserve the orientational order, while more compact stiff nano-rods and especially nano-sheets reduce the order parameter to a larger extent. In spite of the difference between the sizes of the DNA nano-particles and the rod-like columnar aggregates forming the liquid crystal, a similarity between the shapes of the former and the latter seems to be better compatible with the orientational order of the liquid crystal.

DNA-based nanostructures have received great attention as molecular vehicles for cellular delivery of biomolecules and cancer drugs. Here, we report on the cellular uptake of tubule-like DNA tile-assembled nanostructures 27 nm in length and 8 nm in diameter that carry siRNA molecules, folic acid and fluorescent dyes. In our observations, the DNA structures are delivered to the endosome and do not reach the cytosol of the GFP-expressing HeLa cells that were used in the experiments. Consistent with this observation, no elevated silencing of the GFP gene could be detected. Furthermore, the presence of up to six molecules of folic acid on the carrier surface did not alter the uptake behavior and gene silencing. We further observed several challenges that have to be considered when performing in vitro and in vivo experiments with DNA structures: (i) DNA tile tubes consisting of 42 nt-long oligonucleotides and carrying single- or double-stranded extensions degrade within one hour in cell medium at 37 °C, while the same tubes without extensions are stable for up to eight hours. The degradation is caused mainly by the low concentration of divalent ions in the media. The lifetime in cell medium can be increased drastically by employing DNA tiles that are 84 nt long. (ii) Dyes may get cleaved from the oligonucleotides and then accumulate inside the cell close to the mitochondria, which can lead to misinterpretation of data generated by flow cytometry and fluorescence microscopy. (iii) Single-stranded DNA carrying fluorescent dyes are internalized at similar levels as the DNA tile-assembled tubes used here.

DNA nanotechnology offers the possibility to rationally design structures with emergent properties by precisely controlling their geometry and functionality. Here, we demonstrate a DNA-based plasmonic metamolecule that is capable of sensing human thrombin proteins. The chiral reconfigurability of a DNA origami structure carrying two gold nanorods was used to provide optical read-out of thrombin binding through changes in the displayed plasmonic circular dichroism. In our experiments, each arm of the structure was modified with one of two different thrombin-binding aptamers—thrombin-binding aptamer (TBA) and HD22—in such a way that a thrombin molecule could be sandwiched by the aptamers to lock the metamolecule in a state of defined chirality. Our structure exhibited a Kd of 1.4 nM, which was an order of magnitude lower than those of the individual aptamers. The increased sensitivity arose from the avidity gained by the cooperative binding of the two aptamers, which was also reflected by a Hill coefficient of 1.3 ± 0.3. As we further exploited the strong plasmonic circular dichroism (CD) signals of the metamolecule, our method allowed one-step, high sensitivity optical detection of human thrombin proteins in solution.

Owing to their unique optical properties, colloidal quantum dots (QDs) have attracted much attention as versatile fluorescent markers with broad biological and physical applications. On the other hand, DNA-based assembly has proven to be a powerful bottom-up approach to create designer nanoscale objects and to use these objects for the site-directed arrangement of guest components. To achieve good colloidal stability and accurate positioning of QDs on DNA templates, robust QD surface functionalization is crucial. Here, we present a simple and reliable conjugation method for the direct attachment of DNA molecules to QDs. Phosphorothiolated regions of chimera oligonucleotides are attached and incorporated into a ZnS layer freshly growing in situ on QDs that were rendered water soluble with hydrophilic ligands in a prior step. The reaction can be completed in a 2 mL plastic tube without any special equipment. The utility of these DNA-labeled QDs is demonstrated via prototypical assemblies such as QDs dimers with various spacings and chiral helical architectures.

Nanometer-sized polyhedral wire-frame objects hold a wide range of potential applications both as structural scaffolds as well as a basis for synthetic nanocontainers. The utilization of DNA as basic building blocks for such structures allows the exploitation of bottom-up self-assembly in order to achieve molecular programmability through the pairing of complementary bases. In this work, we report on a hollow but rigid tetrahedron framework of 75 nm strut length constructed with the DNA origami method. Flexible hinges at each of their four joints provide a means for structural variability of the object. Through the opening of gaps along the struts, four variants can be created as confirmed by both gel electrophoresis and direct imaging techniques. The intrinsic site addressability provided by this technique allows the unique targeted attachment of dye and/or linker molecules at any point on the structure's surface, which we prove through the superresolution fluorescence microscopy technique DNA PAINT.

In this mini-review recent applications of quantum dots in bioanalytics and biolabeling are discussed. The state-of-the-art of the field is summarized, some selected applications are highlighted, and future directions are suggested. [ABSTRACT FROM AUTHOR]