No abstract

Structural antibody database (SAbDab; http://opig.stats.ox.ac.uk/webapps/sabdab) is an online resource containing all the publicly available antibody structures annotated and presented in a consistent fashion. The data are annotated with several properties including experimental information, gene details, correct heavy and light chain pairings, antigen details and, where available, antibody–antigen binding affinity. The user can select structures, according to these attributes as well as structural properties such as complementarity determining region loop conformation and variable domain orientation. Individual structures, datasets and the complete database can be downloaded.

DNA is typically impermeable to the plasma membrane due to its polyanionic nature. Interestingly, several different DNA nanostructures can be readily taken up by cells in the absence of transfection agents, which suggests new opportunities for constructing intelligent cargo delivery systems from these biocompatible, nonviral DNA nanocarriers. However, the underlying mechanism of entry of the DNA nanostructures into the cells remains unknown. Herein, we investigated the endocytotic internalization and subsequent transport of tetrahedral DNA nanostructures (TDNs) by mammalian cells through single-particle tracking. We found that the TDNs were rapidly internalized by a caveolin-dependent pathway. After endocytosis, the TDNs were transported to the lysosomes in a highly ordered, microtubule-dependent manner. Although the TDNs retained their structural integrity within cells over long time periods, their localization in the lysosomes precludes their use as effective delivery agents. To modulate the cellular fate of the TDNs, we functionalized them with nuclear localization signals that directed their escape from the lysosomes and entry into the cellular nuclei. This study improves our understanding of the entry into cells and transport pathways of DNA nanostructures, and the results can be used as a basis for designing DNA-nanostructure-based drug delivery nanocarriers for targeted therapy.

Patients with acute kidney injury (AKI) frequently require kidney transplantation and supportive therapies, such as rehydration and dialysis. Here, we show that radiolabelled DNA origami nanostructures (DONs) with rectangular, triangular and tubular shapes accumulate preferentially in the kidneys of healthy mice and mice with rhabdomyolysis-induced AKI, and that rectangular DONs have renal-protective properties, with efficacy similar to the antioxidant N-acetylcysteine—a clinically used drug that ameliorates contrast-induced AKI and protects kidney function from nephrotoxic agents. We evaluated the biodistribution of DONs non-invasively via positron emission tomography, and the efficacy of rectangular DONs in the treatment of AKI via dynamic positron emission tomography imaging with 68Ga-EDTA, blood tests and kidney tissue staining. DNA-based nanostructures could become a source of therapeutic agents for the treatment of AKI and other renal diseases.

Self-assembled DNA nanostructures have emerged as a type of nano-biomaterials with precise structures, versatile functions and numerous applications. One particularly promising application of these DNA nanostructures is to develop universal nanocarriers for smart and targeted drug delivery. DNA is the genetic material in nature, and inherently biocompatible. Nevertheless, cell membranes are barely permeable to naked DNA molecules, either single- or double- stranded; transport across the cell membrane is only possible with the assistance of transfection agents. Interestingly, recent studies revealed that many DNA nanostructures could readily go into cells with high cell uptake efficiency. In this Progress Report, we will review recent advances on using various DNA nanostructures, e.g., DNA nanotubes, DNA tetrahedra, and DNA origami nanorobot, as drug delivery nanocarriers, and demonstrate several examples aiming at therapeutic applications with CpG-based immunostimulatory and siRNA-based gene silencing oligonucleotides.

There remains a great challenge in the sensitive detection of microRNA because of the short length and low abundance of microRNAs in cells. Here, we have demonstrated an ultrasensitive detection platform for microRNA by combining the tetrahedral DNA nanostructure probes and hybridization chain reaction (HCR) amplification. The detection limits for DNA and microRNA are 100 aM and 10 aM (corresponding to 600 microRNAs in a 100 μL sample), respectively. Compared to the widely used supersandwich amplification, the detection limits are improved by 3 orders of magnitude. The uncontrolled surface immobilization and consumption of target molecules that limit the amplification efficiency of supersandwich are eliminated in our platform. Taking advantage of DNA nanotechnology, we employed three-dimensional tetrahedral DNA nanostructure as the scaffold to immobilize DNA recognition probes to increase the reactivity and accessibility, while DNA nanowire tentacles are used for efficient signal amplification by capturing multiple catalytic enzymes in a highly ordered way. The synergetic effect of DNA tetrahedron and nanowire tentacles have proven to greatly improve sensitivity for both DNA and microRNA detection.

Self-assembled DNA nanostructures with precise sizes allow a programmable "soft lithography" approach to engineer the interface of electrochemical DNA sensors. By using millimeter-sized gold electrodes modified with several types of tetrahedral DNA nanostructures (TDNs) of different sizes, both the kinetics and thermodynamics of DNA hybridization were profoundly affected. Because each DNA probe is anchored on an individual TDN, its lateral spacing and interactions are finely tuned by the TDN size. By simply varying the size of the TDNs, the hybridization time was decreased and the hybridization efficiency was increased. More significantly, the detection limit for DNA detection was tuned over four orders of magnitude with differentially nanostructured electrodes, and achieved attomolar sensitivity with polymeric enzyme amplification.

The occurrence and prognosis of many complex diseases, such as cancers, is associated with the variation of various molecules, including DNA at the genetic level, RNA at the regulatory level, proteins at the functional level and small molecules at the metabolic level (defined collectively as multilevel molecules). Thus it is highly desirable to develop a single platform for detecting multilevel biomarkers for early-stage diagnosis. Here we report a protocol on DNA-nanostructure-based programmable engineering of the biomolecular recognition interface, which provides a universal electrochemical biosensing platform for the ultrasensitive detection of nucleic acids (DNA/RNA), proteins, small molecules and whole cells. The protocol starts with the synthesis of a series of differentially sized, self-assembled tetrahedral DNA nanostructures (TDNs) with site-specifically modified thiol groups that can be readily anchored on the surface of a gold electrode with high reproducibility. By exploiting the rigid structure, nanoscale addressability and versatile functionality of TDNs, one can tailor the type of biomolecular probes appended on individual TDNs for the detection of specific molecules of interest. Target binding occurring on the gold surface patterned with TDNs is quantitatively translated into electrochemical signals via a coupled enzyme-based catalytic process. This uses a sandwich assay strategy in which biotinylated reporter probes recognize TDN-bound target biomolecules, which then allow binding of horseradish-peroxidase-conjugated avidin (avidin-HRP). Hydrogen peroxide (H2O2) is then reduced by avidin-HRP in the presence of TMB (3,3',5,5'-tetramethylbenzidine) to generate a quantitative electrochemical signal. The time range for the entire protocol is ∼1 d, whereas the detection process takes ∼30 min to 3 h.