Isothermal Hybridization Kinetics of DNA Assembly of Two‐Dimensional DNA Origami

The Watson–Crick base‐pairing with specificity and predictability makes DNA molecules suitable for building versatile nanoscale structures and devices, and the DNA origami method enables researchers to incorporate more complexities into DNA‐based devices. Thermally controlled atomic force microscopy in combination with nanomechanical spectroscopy with forces controlled in the pico Newton (pN) range as a novel technique is introduced to directly investigate the kinetics of multistrand DNA hybridization events on DNA origami nanopores under defined isothermal conditions. For the synthesis of DNA nanostructures under isothermal conditions at 60 °C, a higher hybridization rate, fewer defects, and a higher stability are achieved compared to room‐temperature studies. By quantifying the assembly times for filling pores in origami structures at several constant temperatures, the fill factors show a consistent exponential increase over time. Furthermore, the local hybridization rate can be accelerated by adding a higher concentration of the staples. The new insight gained on the kinetics of staple‐scaffold hybridization on the synthesis of two dimensional DNA origami structures may open up new routes and ideas for designing DNA assembly systems with increased potential for their application.     

Core–Shell and Layer‐by‐Layer Assembly of 3D DNA Crystals

A long‐standing goal of DNA nanotechnology has been to assemble 3D crystals to be used as molecular scaffolds. The DNA 13‐mer, BET66, self‐assembles via Crick–Watson and noncanonical base pairs to form crystals. The crystals contain solvent channels that run through them in multiple directions, allowing them to accommodate tethered guest molecules. Here, the first example of biomacromolecular core–shell crystal growth is described, by showing that these crystals can be assembled with two or more discrete layers. This approach leads to structurally identical layers on the DNA level, but with each layer differentiated based on the presence or absence of conjugated guest molecules. The crystal solvent channels also allow layer‐specific postcrystallization covalent attachment of guest molecules. Through controlling the guest‐molecule identity, concentration, and layer thickness, this study opens up a new method for using DNA to create multifunctional periodic biomaterials with tunable optical, chemical, and physical properties.     

Tunable Confinement for Bridging Single‐Cell Manipulation and Single‐Molecule DNA Linearization

DNA linearization by nanoconfinement has offered a new avenue toward large‐scale genome mapping. The ability to smoothly interface the widely different length scales from cell manipulation to DNA linearization is critical to the development of single‐cell genomic mapping or sequencing technologies. Conventional nanochannel technologies for DNA analysis suffer from complex fabrication procedures, DNA stacking at the nanochannel entrance, and inefficient solution exchange. In this work, a dynamic and tunable confinement strategy is developed to manipulate and linearize genomic‐length DNA molecules from a single cell. By leveraging pneumatic microvalve control and elastomeric collapse, an array of nanochannels with confining dimension down to 20 nm and length up to sub‐millimeter is created and can be dynamically tuned in size. The curved edges of the microvalve form gradual transitions from microscale to nanoscale confinement, smoothly facilitating DNA entry into the nanochannels. A unified micro/nanofluidic device that integrates single‐cell trapping and lysis, DNA extraction, purification, labeling, and linearization is developed based on dynamically controllable nanochannels. Mbp‐long DNA molecules are extracted directly from a single cell and in situ linearized in the nanochannels. The device provides a facile and promising platform to achieve the ultimate goal of single‐cell, single‐genome analysis.     

Programmable DNA Nanoindicator‐Based Platform for Large‐Scale Square Root Logic Biocomputing

The prospect of programming molecular computing systems to realize complex autonomous tasks has advanced the design of synthetic biochemical logic circuits. One way to implement digital and analog integrated circuits is to use noncovalent hybridization and strand displacement reactions in cell‐free and enzyme‐free nucleic acid systems. To date, DNA‐based circuits involving tens of logic gates capable of implementing basic and complex logic functions have been demonstrated experimentally. However, most of these circuits are still incapable of realizing complex mathematical operations, such as square root logic operations, which can only be carried out with 4 bit binary numbers. A high‐capacity DNA biocomputing system is demonstrated through the development of a 10 bit square root logic circuit. It can calculate the square root of a 10 bit binary number (within the decimal integer 900) by designing DNA sequences and programming DNA strand displacement reactions. The input signals are optimized through the output feedback to improve performance in more complex logical operations. This study provides a more universal approach for applications in biotechnology and bioengineering.     

Daunorubicin‐Loaded DNA Origami Nanostructures Circumvent Drug‐Resistance Mechanisms in a Leukemia Model

Many cancers show primary or acquired drug resistance due to the overexpression of efflux pumps. A novel mechanism to circumvent this is to integrate drugs, such as anthracycline antibiotics, with nanoparticle delivery vehicles that can bypass intrinsic tumor drug‐resistance mechanisms. DNA nanoparticles serve as an efficient binding platform for intercalating drugs (e.g., anthracyclines doxorubicin and daunorubicin, which are widely used to treat acute leukemias) and enable precise structure design and chemical modifications, for example, for incorporating targeting capabilities. Here, DNA nanostructures are utilized to circumvent daunorubicin drug resistance at clinically relevant doses in a leukemia cell line model. The fabrication of a rod‐like DNA origami drug carrier is reported that can be controllably loaded with daunorubicin. It is further directly verified that nanostructure‐mediated daunorubicin delivery leads to increased drug entry and retention in cells relative to free daunorubicin at equal concentrations, which yields significantly enhanced drug efficacy. Our results indicate that DNA origami nanostructures can circumvent efflux‐pump‐mediated drug resistance in leukemia cells at clinically relevant drug concentrations and provide a robust DNA nanostructure design that could be implemented in a wide range of cellular applications due to its remarkably fast self‐assembly (≈5 min) and excellent stability in cell culture conditions.     

Stimuli‐Responsive Self‐Assembled DNA Nanomaterials for Biomedical Applications

Stimuli‐responsive DNA‐based materials represent a major class of remarkable functional nanomaterials for nano‐biotechnological applications. In this review, recent progress in the development of stimuli‐responsive systems based on self‐assembled DNA nanostructures is introduced and classified. Representative examples are presented in terms of their design, working principles and mechanisms to trigger the response of the stimuli‐responsive DNA system upon expose to a large variety of stimuli including pH, metal ions, oligonucleotides, small molecules, enzymes, heat, and light. Substantial in vitro studies have clearly revealed the advantages of the use of stimuli‐responsive DNA nanomaterials in different biomedical applications, particularly for biosensing, drug delivery, therapy and diagnostic purposes in addition to bio‐computing. Some of the challenges faced and suggestions for further development are also highlighted.     

Solution‐Controlled Conformational Switching of an Anchored Wireframe DNA Nanostructure

Self‐assembled DNA origami nanostructures have a high degree of programmable spatial control that enables nanoscale molecular manipulations. A surface‐tethered, flexible DNA nanomesh is reported herein which spontaneously undergoes sharp, dynamic conformational transitions under physiological conditions. The transitions occur between two major macrostates: a spread state dominated by the interaction between the DNA nanomesh and the BSA/streptavidin surface and a surface‐avoiding contracted state. Due to a slow rate of stochastic transition events on the order of tens of minutes, the dynamic conformations of individual structures can be detected in situ with DNA PAINT microscopy. Time series localization data with automated imaging processing to track the dynamically changing radial distribution of structural markers are combined. Conformational distributions of tethered structures in buffers with elevated pH exhibit a calcium‐dependent domination of the spread state. This is likely due to electrostatic interactions between the structures and immobilized surface proteins (BSA and streptavidin). An interaction is observed in solution under similar buffer conditions with dynamic light scattering. Exchanging between solutions that promote one or the other state leads to in situ sample‐wide transitions between the states. The technique herein can be a useful tool for dynamic control and observation of nanoscale interactions and spatial relationships.     

Surface Modification of Solid‐State Nanopores for Sticky‐Free Translocation of Single‐Stranded DNA

Nanopore technology is one of the most promising approaches for fast and low‐cost DNA sequencing application. Single‐stranded DNA detection is primary objective in such device, while solid‐state nanopores remain less explored than their biological counterparts due to bio‐molecule clogging issue caused by surface interaction between DNA and nanopore wall. By surface coating a layer of polyethylene glycol (PEG), solid‐state nanopore can achieve long lifetime for single‐stranded DNA sticky‐free translocation at pH 11.5. Associated with elimination of non‐specific binding of molecule, PEG coated nanopore presents new surface characteristic as less hydrophility, lower 1/f noise, and passivated surface charge responsiveness on pH. Meanwhile, conductance blockage of single‐stranded DNA is found to be deeper than double‐stranded DNA, which can be well described by a string of blobs model for a quasi‐equilibrium state polymer in constraint space.