Modular Weaving DNAzyme in Skeleton of DNA Nanocages for Photoactivatable Catalytic Activity Regulation.

DNAzymes exhibit tremendous application potentials in the field of biosensing and gene regulation due to its unique catalytic function. However, spatiotemporally controlled regulation of DNAzyme activity remains a daunting challenge, which may cause nonspecific signal leakage or gene silencing of the catalytic systems. Here, we report a photochemical approach via modular weaving active DNAzyme into the skeleton of tetrahedral DNA nanocages (TDN) for light-triggered on-demand liberation of DNAzyme and thus conditional control of gene regulation activity. We demonstrate that the direct encoding of DNAzyme in TDN could improve the biostability of DNAzyme and ensure the delivery efficiency, comparing with the conventional surface anchoring strategy. Furthermore, the molecular weaving of the DNA nanostructures allows remote control of DNAzyme-mediated gene regulation with high spatiotemporal precision of light. In addition, we demonstrate that the approach is applicable for controlled regulation of the gene editing functions of other functional nucleic acids.

Modular Engineering of DNAzyme-Based Sensors for Spatioselective Imaging of Metal Ions in Mitochondria.

DNAzyme-based sensors remain at the forefront of metal-ion imaging efforts, but most lack the subcellular precision necessary to their applications in specific organelles. Here, we seek to overcome this limitation by presenting a DNAzyme-based biosensor technology for spatiotemporally controlled imaging of metal ions in mitochondria. A DNA nanodevice was constructed by integrating an optically activatable DNAzyme sensor and an upconversion nanoparticle with an organelle-targeting signal. We exemplify that this approach allows for mitochondria-specific imaging of Zn2+ in living cells in a near-infrared light-controlled manner. Based on this, the system is used for the monitoring of mitochondrial Zn2+ during drug treatment in a cellular model of ischemia insult. Furthermore, the DNA nanodevice is employed to assess dynamic Zn2+ change and pharmacological interventions in an injury cell model of Zn2+ toxicity. This method paves the way for engineering of DNAzyme sensors to investigate the pathophysiological roles of metal ions at the subcellular level.

A Remotely Controlled Nanosystem for Spatiotemporally Specific Gene Regulation and Combinational Tumor Therapy.

The dearth of technologies that allow gene modulation and therapy with high spatiotemporal precision remains a bottleneck in biomedical research and applications. Here we present a near-infrared (NIR) light-controlled nanosystem that allows spatiotemporally controlled regulation of gene expression and thus combinational tumor therapy. The nanosystem is built by engineering of an enzyme-activatable antisense oligonucleotide and further combination with an upconversion nanoparticle-based photodynamic system and a mitochondria localization signal. The system relies on photodynamic effect-induced translocation of a DNA repair enzyme from nucleus into mitochondria, which enables spatially selective gene regulation via enzymatic reactions. We demonstrate that the NIR light-induced mitochondrial photodamage and gene regulation enable enhanced antitumor effect. Our approach may enable the specific gene regulation and tumor treatment with high precision both spatially and temporally.

An Enzyme-Activatable Engineered DNAzyme Sensor for Cell-Selective Imaging of Metal Ions.

There is intense interest in imaging intracellular metal ions because of their vital roles in many cellular processes. The challenge has been to develop chemical approaches that can detect metal ions in a cell-type-specific manner. Herein we report the design of an enzyme-activatable DNAzyme sensor technology that can distinguish metal-ion signals in tumor cells from those in normal cells both in vitro and in vivo. Specifically, the sensing activity of a traditional DNAzyme sensor was inhibited by engineering with a blocking sequence containing an abasic site that can be cleaved by cancer-specific enzymes and thus enables the selective recovery of metal-ion sensing capability in cancer cells. We demonstrated that the DNAzyme sensor not only enables cancer-cell-selective sensing and imaging of metal ions through an enzymically activated pathway, but also precise control over its metal-ion sensing activity in tumor-bearing mice. We envision the use of this biosensing technology to probe the biological roles of diverse metal ions in specific diseases.

Peptide Nucleic Acid (PNA)-Guided Peptide Engineering of an Aptamer Sensor for Protease-Triggered Molecular Imaging.

Protease-triggered control of functional DNA has remained unachieved, leaving a significant gap in activatable DNA biotechnology. Herein, we report the design of a protease-activatable aptamer system that can perform molecular sensing and imaging in a tumor-specific manner. The system is constructed by locking the structure-switching activity of an aptamer using a rationally designed PNA-peptide-PNA triblock copolymer. Highly selective protease-mediated cleavage of the peptide substrate results in reduced binding affinity of PNA to the aptamer module, with the subsequent recovery of its biosensing function. We demonstrated that the DNA/peptide/PNA hybrid system allows for tumor cell-selective ATP imaging in vitro and also produces a fluorescent signal in vivo with improved tumor specificity. This work illustrates the potential of bridging the gap between functional DNA and peptides for precise biomedical applications.