Jiye Shi

Hybridization Chain Reaction Amplification of MicroRNA Detection with a Tetrahedral DNA Nanostructure-Based Electrochemical Biosensor

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.

Smart Drug Delivery Nanocarriers with Self-Assembled DNA Nanostructures

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.

Single-Particle Tracking and Modulation of Cell Entry Pathways of a Tetrahedral DNA Nanostructure in Live Cells

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.

Electrochemical detection of nucleic acids, proteins, small molecules and cells using a DNA-nanostructure-based universal biosensing platform

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.

DNA-Directed Assembly of Gold Nanohalo for Quantitative Plasmonic Imaging of Single-Particle Catalysis

Plasmonic imaging under a dark-field microscope (DFM) holds great promise for single-particle analysis in bioimaging, nanophotonics, and nanocatalysis. Here, we designed a DNA-directed programmable assembly strategy to fabricate a halo-like Au nanostructure (nanohalo) that couples plasmonic large gold nanoparticles (L-AuNPs) with catalytically active small AuNPs (S-AuNPs) in a single nanoarchitecture. Catalytic reaction occurring on S-AuNPs changes its permittivity, which results in a significant variation of the plasmonic resonance of the nanohalo. Hence, we can indirectly monitor catalytic reactions on a single nanohalo under DFM, on the basis of which we have obtained quantitative information on both nanocatalysis and catalyst poisoning. Our study thus provides a cost-effective means to quantitatively study metal NP-based catalysis at single-particle level.

An Exonuclease III‐Powered, On‐Particle Stochastic DNA Walker

DNA-based machines have attracted rapidly growing interest owing to their potential in drug delivery, biocomputing, and diagnostic applications. Herein, we report a type of exonuclease III (Exo III)-powered stochastic DNA walker that can autonomously move on a spherical nucleic acid (SNA)-based 3D track. The motion is propelled by unidirectional Exo III digestion of hybridized DNA tracks in a burnt-bridge mechanism. The operation of this Exo III-propelled DNA walker was monitored in real time and at the single-particle resolution using total internal reflection fluorescence microscopy (TIRF). We further interrogated the morphological effect of the 3D track on the nuclease activity, which suggested that the performance of the DNA walker was critically dependent upon the DNA density and the track conformation. Finally, we demonstrated potential bioanalytical applications of this SNA-based stochastic DNA walker by exploiting movement-triggered cascade signal amplification.

Gold-Nanoparticle-Mediated Jigsaw-Puzzle-like Assembly of Supersized Plasmonic DNA Origami

DNA origami has rapidly emerged as a powerful and programmable method to construct functional nanostructures. However, the size limitation of approximately 100 nm in classic DNA origami hampers its plasmonic applications. Herein, we report a jigsaw-puzzle-like assembly strategy mediated by gold nanoparticles (AuNPs) to break the size limitation of DNA origami. We demonstrated that oligonucleotide-functionalized AuNPs function as universal joint units for the one-pot assembly of parent DNA origami of triangular shape to form sub-microscale super-origami nanostructures. AuNPs anchored at predefined positions of the super-origami exhibited strong interparticle plasmonic coupling. This AuNP-mediated strategy offers new opportunities to drive macroscopic self-assembly and to fabricate well-defined nanophotonic materials and devices.

Self-Assembly of Poly-Adenine-Tailed CpG Oligonucleotide-Gold Nanoparticle Nanoconjugates with Immunostimulatory Activity

Synthetic unmethylated cytosine-guanine (CpG) oligodeoxynucleotides (CpG ODNs) possess high immunostimulatory activity and have been widely used as a therapeutic tool for various diseases including infection, allergies, and cancer. A variety of nanocarriers have been developed for intracellular delivery of CpG ODNs that are otherwise nonpermeable through the cellular membrane. For example, previous studies showed that gold nanoparticles (AuNPs) could efficiently deliver synthetic thiolated CpG ODNs into cultured cells and induce expression of proinflammatory cytokines. Nevertheless, the necessity of using thiolated CpG ODNs for the modification of AuNPs inevitably complicates the synthesis of the nanoconjugates and increases the cost. A new approach is demonstrated for facile assembly of AuNP-CpG nanoconjugates for cost-effective drug delivery. It is found that non-thiolated, diblock ODNs containing a CpG motif and a poly-adenine (polyA) tail can readily self-assemble on the surface of AuNPs with controllable and tunable density. Such nanoconjugates are efficiently delivered into RAW264.7 cells and induce immune response in a Toll-like receptor 9 (TLR9)-dependent manner. Under optimal conditions, polyA-CpG-AuNPs show significantly higher immunostimulatory activity than their thiolated counterpart. In addition, the immunostimulatory activity of CpG-AuNPs can be modulated by varying the length of the polyA tail. In vivo induction of immune responses in mice is demonstrated by using polyA-tailed CpG-AuNP nanoconjugates.

Size-dependent programming of the dynamic range of graphene oxide-DNA interaction-based ion sensors.

Graphene oxide (GO) is widely used in biosensors and bioimaging because of its high quenching efficiency, facile chemical conjugation, unique amphiphile property, and low cost for preparation. However, the nanometer size effect of GO on GO-DNA interaction has long been ignored and remains unknown. Here we examined the nanometer size effect of GO on GO-DNA interactions. We concluded that GO of ∼200 nm (lateral nanometer size) possessed the highest fluorescence quenching efficiency whereas GO of ∼40 nm demonstrated much weaker ability to quench the fluorescence. We employed the nanometer size effect of GO to program the dynamic ranges and sensitivity of mercury sensors. Three dynamic ranges (1 to 40 nM, 1 to 15 nM, and 0.1 to 5 nM) were obtained with this size modulation. The sensitivity (slope of titration curve) was programmed from 15.3 ± 1.27 nM(-1) to 106.2 ± 3.96 nM(-1).

One-Shot Immunomodulatory Nanodiamond Agents for Cancer Immunotherapy.

The use of functional nanodiamonds (fNDs) to deliver CpG oligonucleotides (ODNs) for sustained immunostimulation is reported. It is demonstrated that monotherapy using this immunostimulatory agent significantly suppresses the tumor growth in two murine tumor models. This fND-based nanoagent opens new opportunities for immunotherapy, as well as clinical applications of various types of therapeutic nucleic acids.