Precision-Guided Missile-Like DNA Nanostructure Containing Warhead and Guidance Control for Aptamer-Based Targeted Drug Delivery into Cancer Cells in Vitro and in Vivo.

It is crucial to deliver anticancer drugs to target cells with high precision and efficiency. While nanomaterials have been shown to enhance the delivery efficiency once they reach the target, it remains challenging for precise drug delivery to overcome the nonspecific adsorption and off-target effect. To meet this challenge, we report herein the design of a novel DNA nanostructure to act as a DNA nanoscale precision-guided missile (D-PGM) for highly efficient loading and precise delivery of chemotherapeutic agents to specific target cells. The D-PGM consists of two parts: a warhead (WH) and a guidance/control (GC). The WH is a rod-like DNA nanostructure as a drug carrier, whose trunk is a three-dimensionally self-assembled DNA nanoscale architecture from the programmed hybridization among two palindromic DNA sequences in the x-y dimension and two common DNA oligonucleotides in the z direction, making the WH possess a high payload capacity of drugs. The GC is an aptamer-based logic gate assembled in a highly organized fashion capable of performing cell-subtype-specific recognition via the sequential disassembly, mediated by cell-anchored aptamers. Because of the cooperative effects between the WH and the GC, the GC logic gates operate like the guidance and control system in a precision-guided missile to steer the doxorubicin (DOX)-loaded DNA WH toward target cancer cells, leading to selective and enhanced therapeutic efficacy. Moreover, fluorophores attached to different locations of D-PGM and DOX fluorescence dequenching upon release enable intracellular tracing of the DNA nanostructures and drugs. The results demonstrate that by mimicking the functionalities of a military precision-guided missile to design the sequential disassembly of the GC system in multistimuli-responsive fashion, our intrinsically biocompatible and degradable D-PGM can accurately identify target cancer cells in complex biological milieu and achieve active targeted drug delivery. The success of this strategy paves the way for specific cell identity and targeted cancer therapy.

Y-Shaped Backbone-Rigidified Triangular DNA Scaffold-Directed Stepwise Movement of a DNAzyme Walker for Sensitive MicroRNA Imaging within Living Cells.

DNA as a programmable molecule shows great potential in a wide variety of applications, with the dynamic DNA nanodevices such as DNA motors and walkers holding the most promise in controlled functions for biosensing and nanomedicine. However, a motor or walker that consists of DNA exclusively has not been shown to function within cells because of its susceptibility to endogenous nuclease-mediated degradation. In this contribution, we demonstrate a Y-shaped backbone-rigidified triangular DNA scaffold (YTDS)-directed DNAzyme walker that functions inside living cells to detect microRNAs (miRNAs) with high sensitivity. A novel Y-shaped backbone offers access to geometrically well-defined configurations and increases the rigidity of DNA assemblies, providing a unique, circular, and rigid DNA track within living cells without non-nucleic acid auxiliary materials and enabling the stepwise movement of DNAzyme in an inchworm fashion. This strategy is extended to the construction of larger rigid planar geometric polygon-based DNA walkers, demonstrating unprecedented opportunities to build dynamic DNA nanostructures with precise geometry and versatile functionality.

Mitochondria and Nuclei Dual-Targeted Hollow Carbon Nanospheres for Cancer Chemophotodynamic Synergistic Therapy.

Dual-targeted nanoparticles are gaining increasing importance as a more effective anticancer strategy by attacking double key sites of tumor cells, especially in chemophotodynamic therapy. To retain the nuclei inhibition effect and enhance doxorubicin (DOX)-induced apoptosis by mitochondrial pathways simultaneously, we synthesized the novel nanocarrier (HKH) based on hollow carbon nitride nanosphere (HCNS) modified with hyaluronic acid (HA) and the mitochondrial localizing peptide D[KLAKLAK]2 (KLA). DOX-loaded HKH nanoparticles (HKHDs) showed satisfactory drug-loading efficiency, excellent solubility, and very low hemolytic effect. HA/CD44 binding and electrostatic attraction between positively charged KLA and A549 cells facilitated HKHD uptake via the endocytosis mechanism. Acidic microenvironment, hyaluronidase, and KLA targeting together facilitate doxorubicin toward the mitochondria and nuclei, resulting in apoptosis, DNA intercalation, cell-cycle arrest at the S phase, and light-induced reactive oxygen species production. Intravascular HKHD inhibited tumor growth in A549-implanted mice with good safety. The present study, for the first time, systemically reveals biostability, targetability, chemophotodynamics, and safety of the functionalized novel HKHD.

Inverted mirror image molecular beacon-based three concatenated logic gates to detect p53 tumor suppressor gene.

Mutation of p53 tumor suppressor gene represents one of the early molecular events in tumor initiation and progression. Although molecular computing holds tremendous potential with important applications in diagnosis, prognosis and treatment of human diseases at the molecular level, designing molecular logic gates to implement cascade amplification via operating autonomously for the detection of point mutations still remains challenging. In this contribution, we developed a three concatenated logic gates (TCLG) to perform multiple strand displacement amplification (m-SDA) for screening the cancer-related point mutations only via designing an innovative molecular beacon (MB). Specifically, using p53 gene as model target, extending the two ends of a MB via adding two fragments with the same sequence achieves two unique terminal single-stranded (ss) overhangs. After self-folding of MB into hairpin structure, the two overhangs exhibit a near inverted mirror image (IM) relationship if taking the base nature and direction into account. For this, the probe is called IM-MB. Because cascade SDAs can occur on IM-MB and promote each other, the target gene can be detected down to 10 pM. Along this line, the TCLG circuit was proposed, and two primers and target gene serve as the indispensable input signals. Utilizing this logic circuit, the point mutation or absence of target gene can be sensitively screened. Moreover, its potential application in the recognition of point mutations in complex biomatrix has been demonstrated via blind test. The proof-of-concept scheme is expected to provide new insight into the development of DNA-based molecular logic gates and their applications in basic research, medical diagnosis and precise treatment and treatment of genetic diseases.

DNA nanostructures from palindromic rolling circle amplification for the fluorescent detection of cancer-related microRNAs.

Herein, DNA nanostructures were prepared via a palindromic padlock probe-based rolling circle amplification (called P-RCA) and then employed to implement the sensitive and specific detection of let-7a miRNA extracted from cancer cells without chemical modification. The presence of target let-7a miRNA as a polymerization primer can trigger the P-RCA process, generating a long tandemly repetitive DNA strand. The resulting products can fold into nanostructures via self-hybridization of palindromic regions and possess numerous double-stranded fragments. In this case, the strong fluorescent signal is detected upon exposure to SYBR Green I. As a result, in homogeneous solution, target miRNA can be detected down to 6.4 pM with a wide dynamic range. A high specificity was demonstrated by the excellent discrimination between let-7 miRNA family members, while the applicability of this sensing system in complex biological environments was confirmed by the analysis of target miRNAs extracted from HeLa cells. It should be noted that increasing numbers of palindromic fragments in padlock probe further increases signal amplification efficiency. The experimental results indicate that the newly proposed P-RCA DNA nanostructures have potential to become a promising analytical platform in biomedical research and clinical diagnosis for the miRNA detection with high sensitivity and good specificity.

Target-Induced Catalytic Assembly of Y-Shaped DNA and Its Application for In†Situ Imaging of MicroRNAs.

DNA is a highly programmable material that can be configured into unique high-order structures, such as DNA branched junctions containing multiple helical arms converging at a center. Herein we show that DNA programmability can deliver in situ growth of a 3-way junction-based DNA structure (denoted Y-shaped DNA) with the use of three hairpin-shaped DNA molecules as precursors, a specific microRNA target as a recyclable trigger, and a DNA polymerase as a driver. We demonstrate that the Y-shaped configuration comes with the benefit of restricted freedom of movement in confined cellular environment, which makes the approach ideally suited for in situ imaging of small RNA targets, such as microRNAs. Comparative analysis illustrates that the proposed imaging technique is superior to both the classic fluorescence in situ hybridization (FISH) method and an analogous amplified imaging method via programmed growth of a double-stranded DNA (rather than Y-shaped DNA) product.