Scaffolded DNA origami, a method to create self-assembled nanostructures with spatially addressable features, has recently been used to develop water-soluble molecular chips for label-free RNA detection, platforms for deterministic protein positioning, and single molecule reaction observatories. These applications highlight the possibility of exploiting the unique properties and biocompatibility of DNA nanostructures in live, cellular systems. Herein, we assembled several DNA origami nanostructures of differing shape, size and probes, and investigated their interaction with lysate obtained from various normal and cancerous cell lines. We separated and analyzed the origami–lysate mixtures using agarose gel electrophoresis and recovered the DNA structures for functional assay and subsequent microscopic examination. Our results demonstrate that DNA origami nanostructures are stable in cell lysate and can be easily separated from lysate mixtures, in contrast to natural, single- and double-stranded DNA. Atomic force microscope (AFM) and transmission electron microscope (TEM) images show that the DNA origami structures are fully intact after separation from cell lysates and hybridize to their targets, verifying the superior structural integrity and functionality of self-assembled DNA origami nanostructures relative to conventional oligonucleotides. The stability and functionality of DNA origami structures in cell lysate validate their use for biological applications, for example, as programmable molecular rafts or disease detection platforms.

Understanding the thermodynamic properties of complex DNA nanostructures, including rationally designed two- and three-dimensional (2D and 3D, respectively) DNA origami, facilitates more accurate spatiotemporal control and effective functionalization of the structures by other elements. In this work fluorescein and tetramethylrhodamine (TAMRA), a Förster resonance energy transfer (FRET) dye pair, were incorporated into selected staples within various 2D and 3D DNA origami structures. We monitored the temperature-dependent changes in FRET efficiency that occurred as the dye-labeled structures were annealed and melted and subsequently extracted information about the associative and dissociative behavior of the origami. In particular, we examined the effects of local and long-range structural defects (omitted staple strands) on the thermal stability of common DNA origami structures. The results revealed a significant decrease in thermal stability of the structures in the vicinity of the defects, in contrast to the negligible long-range effects that were observed. Furthermore, we probed the global assembly and disassembly processes by comparing the thermal behavior of the FRET pair at several different positions. We demonstrated that the staple strands located in different areas of the structure all exhibit highly cooperative hybridization but have distinguishable melting temperatures depending on their positions. This work underscores the importance of understanding fundamental aspects of the self-assembly of DNA nanostructures and can be used to guide the design of more complicated DNA nanostructures, to optimize annealing protocol and manipulate functionalized DNA nanostructures.

We demonstrate the synthesis of near-IR-emitting zinc blende CdTe/CdS tetrahedral-shaped nanocrystals with a magic-sized (approximately 0.8 nm radius) CdTe core and a thick CdS shell (up to 5 nm). These high-quality water-soluble nanocrystals were obtained by a simple but reliable aqueous method at low temperature. During the growth of the shell over the magic core, the core/shell nanocrystals change from type I to type II, as revealed by their enormous photoluminescence (PL) emission peak shift (from 480 to 820 nm) and significant increase in PL lifetime (from approximately 1 to approximately 245 ns). These thick-shell nanocrystals have a high PL quantum yield, high photostability, compact size (hydrodynamic diameter less than 11.0 nm), and reduced blinking behavior. The magic-core/thick-shell nanocrystals may represent an important step toward the synthesis and application of next-generation colloidal nanocrystals from solar cell conversion to intracellular imaging.

We describe the use of a frame-guided assembly (FGA) strategy to construct cuboid and dumbbell-shaped hetero-vesicles on DNA origami nanostructure scaffolds. These are achieved by varying the design of the DNA origami scaffolds that direct the distribution of the leading hydrophobic groups (LHG). By careful selection of LHGs, different types of amphiphiles (both polymer and small-molecule surfactants) were guided to form hetero-vesicles, demonstrating the versatility of the FGA strategy and its potential to construct asymmetric and dynamic hetero-vesicle assemblies with complex DNA nano-scaffolds.

CONSPECTUS: DNA nanotechnology is one of the most flourishing interdisciplinary research fields. DNA nanostructures can be designed to self-assemble into a variety of periodic or aperiodic patterns of different shapes and length scales. They can be used as scaffolds for organizing other nanoparticles, proteins, and chemical groups, leveraging their functions for creating complex bioinspired materials that may serve as smart drug delivery systems, in vitro or in vivo biomolecular computing platforms, and diagnostic devices. Achieving optimal structural features, efficient assembly protocols, and precise functional group positioning and modification requires a thorough understanding of the thermodynamics and kinetics of the DNA nanostructure self-assembly process. The most common real-time measurement strategies include monitoring changes in UV absorbance based on the hyperchromic effect of DNA, and the emission signal changes of DNA intercalating dyes or covalently conjugated fluorescent dyes/pairs that accompany temperature dependent structural changes. Thermodynamic studies of a variety of DNA nanostructures have been performed, from simple double stranded DNA formation to more complex origami assembly. The key parameters that have been evaluated in terms of stability and cooperativity include the overall dimensions, the folding path of the scaffold, crossover and nick point arrangement, length and sequence of single strands, and salt and ion concentrations. DNA tile-tile interactions through sticky end hybridization have also been analyzed, and the steric inhibition and rigidity of tiles turn out to be important factors. Many kinetic studies have also been reported, and most are based on double stranded DNA formation. A two-state assumption and the hypothesis of several intermediate states have been applied to determine the rate constant and activation energy of the DNA hybridization process. A few simulated models were proposed to represent the structural, mechanical, and kinetic properties of DNA hybridization. The kinetics of strand displacement reactions has also been studied as a special case of DNA hybridization. The thermodynamic and kinetic characteristics of DNA nanostructures have been exploited to develop rapid and isothermal annealing protocols. It is conceivable that a more thorough understanding of the DNA assembly process could be used to guide the structural design process and optimize the conditions for assembly, manipulation, and functionalization, thus benefiting both upstream design and downstream applications.

Technologies capable of characterizing the full breadth of cellular systems need to be able to measure millions of proteins, isoforms, and complexes simultaneously. We describe an approach that fulfils this criterion: Adaptive Dynamic Artificial Poly-ligand Targeting (ADAPT). ADAPT employs an enriched library of single-stranded oligodeoxynucleotides (ssODNs) to profile complex biological samples, thus achieving an unprecedented coverage of system-wide, native biomolecules. We used ADAPT as a highly specific profiling tool that distinguishes women with or without breast cancer based on circulating exosomes in their blood. To develop ADAPT, we enriched a library of ~1011 ssODNs for those associating with exosomes from breast cancer patients or controls. The resulting 106 enriched ssODNs were then profiled against plasma from independent groups of healthy and breast cancer-positive women. ssODN-mediated affinity purification and mass spectrometry identified low-abundance exosome-associated proteins and protein complexes, some with known significance in both normal homeostasis and disease. Sequencing of the recovered ssODNs provided quantitative measures that were used to build highly accurate multi-analyte signatures for patient classification. Probing plasma from 500 subjects with a smaller subset of 2000 resynthesized ssODNs stratified healthy, breast biopsy-negative, and -positive women. An AUC of 0.73 was obtained when comparing healthy donors with biopsy-positive patients.

Assessing the phenotypic diversity underlying tumour progression requires the identification of variations in the respective molecular interaction networks. Here we report proof-of-concept for a platform called poly-ligand profiling (PLP) that surveys these system states and distinguishes breast cancer patients who did or did not derive benefit from trastuzumab. We perform tissue-SELEX on breast cancer specimens to enrich single-stranded DNA (ssDNA) libraries that preferentially interact with molecular components associated with the two clinical phenotypes. Testing of independent sample sets verifies the ability of PLP to classify trastuzumab-treated patients according to their clinical outcomes with ROC-AUC of 0.78. Standard HER2 testing of the same patients gives a ROC-AUC of 0.47. Kaplan–Meier analysis reveals a median increase in benefit from trastuzumab-containing treatments of 300 days for PLP-positive compared to PLP-negative patients. If prospectively validated, PLP may increase success rates in precision oncology and clinical trials, thus improving both patient care and drug development.

A conductive nanoporous antimony-doped tin oxide (ATO) powder has been prepared using the sol-gel method that contains three-dimensionally interconnected pores within the metal oxide and highly tunable pore sizes on the nanoscale. It is demonstrated that these porous materials possess the capability of hosting a tetrahedral-shaped DNA nanostructure of defined dimensions with high affinity. The tunability of pore size enables the porous substrate to selectively absorb the DNA nanostructures into the metal oxide cavities or exclude them from entering the surface layer. Both confocal fluorescence microscopy and solution FRET experiments revealed that the DNA nanostructures maintained their integrity upon the size-selective incorporation into the cavities of the porous materials. As DNA nanostructures can serve as a stable three-dimensional nanoscaffold for the coordination of electron transfer mediators, this work opens up new possibilities of incorporating functionalized DNA architectures as guest molecules to nanoporous conductive metal oxides for applications such as photovoltaics, sensors, and solar fuel cells.