DNAzyme Delivery Research Articles

Construction of a Stimuli-Responsive DNAzyme-Braked DNA Nanomachine for the Amplified Imaging of miRNAs in Living Cells and Mice

Construction of a Stimuli-Responsive DNAzyme-Braked DNA Nanomachine for the Amplified Imaging of miRNAs in Living Cells and Mice

Metal-Organic Framework-Loaded Engineering DNAzyme for the Self-Powered Amplified Detection of MicroRNA.

DNAzyme shows great promise in designing a highly sensitive and specific sensing platform; however, the low cellular uptake efficiency, instability, and especially the insufficient cofactor supply inhibit the intracellular molecule sensor applications. Herein, we demonstrate a novel type of DNAzyme-based self-driven intracellular sensor for microRNA (miRNA) detection in living cells. The sensor consists of a metal-organic framework [zeolite imidazole framework (ZIF-8)] core loaded with a shell consisting of a rationally designed DNAzyme, where the substrate strand is modified with FAM and BHQ-1 nearby both the sides of the restriction site, respectively, while the enzyme strand consists of two separate strands with a complementary fragment to the substrate strand and the targeting miRNA, respectively. The ZIF-8 nanoparticles enable the efficient delivery of DNAzyme into the cell and protect the DNAzyme from degradation. The pH-responsive ZIF-8 degradation is accompanied with the release of the DNAzyme and Zn2+ cofactors, and the intracellular target miRNAs recognize and activate the DNAzyme driven by the Zn2+ cofactors to cleave the substrate strand, resulting in the release of the FAM-labeled shorter product strand and increased fluorescence for miRNA detection. The self-driven approach can be generally applied to various miRNAs' detection through DNAzyme engineering.

Upconversion Luminescence‐Boosted Escape of DNAzyme from Endosomes for Enhanced Gene‐Silencing Efficacy

AbstractDespite the enormous potential of DNAzyme for gene therapy, its efficacy is hampered by the limited endosomal escape capability. Here, we develop a near‐infrared (NIR) light‐controlled DNAzyme delivery platform to achieve enhanced gene‐silencing efficacy. The nanoplatform is composed of therapeutic DNAzyme, photosensitizers (PSs) and upconversion nanoparticles (UCNPs) that can convert NIR light to visible light. The system allows NIR light‐activatable generation of cytotoxic reactive oxygen species due to the energy transfer from the UCNPs to PSs, which boosts the endosomal escape of DNAzyme for an improved gene‐silencing efficacy. We demonstrate that the nanocomposites represent a promising platform to integrate DNAzyme‐based gene therapy with NIR light‐triggered photodynamic therapy for combinational tumor treatment. This work highlights a robust approach to combat the current limitations of DNAzyme delivery systems.

Upconversion Luminescence-Boosted Escape of DNAzyme from Endosomes for Enhanced Gene-Silencing Efficacy.

Despite the enormous potential of DNAzyme for gene therapy, its efficacy is hampered by the limited endosomal escape capability. Here, we develop a near-infrared (NIR) light-controlled DNAzyme delivery platform to achieve enhanced gene-silencing efficacy. The nanoplatform is composed of therapeutic DNAzyme, photosensitizers (PSs) and upconversion nanoparticles (UCNPs) that can convert NIR light to visible light. The system allows NIR light-activatable generation of cytotoxic reactive oxygen species due to the energy transfer from the UCNPs to PSs, which boosts the endosomal escape of DNAzyme for an improved gene-silencing efficacy. We demonstrate that the nanocomposites represent a promising platform to integrate DNAzyme-based gene therapy with NIR light-triggered photodynamic therapy for combinational tumor treatment. This work highlights a robust approach to combat the current limitations of DNAzyme delivery systems.

DNAzymes, Novel Therapeutic Agents in Cancer Therapy: A Review of Concepts to Applications.

The past few decades have witnessed a rapid evolution in cancer drug research which is aimed at developing active biological interventions to regulate cancer-specific molecular targets. Nucleic acid-based therapeutics, including ribozymes, antisense oligonucleotides, small interference RNA (siRNA), aptamer, and DNAzymes, have emerged as promising candidates regulating cancer-specific genes at either the transcriptional or posttranscriptional level. Gene-specific catalytic DNA molecules, or DNAzymes, have shown promise as a therapeutic intervention against cancer in various in vitro and in vivo models, expediting towards clinical applications. DNAzymes are single-stranded catalytic DNA that has not been observed in nature, and they are synthesized through in vitro selection processes from a large pool of random DNA libraries. The intrinsic properties of DNAzymes like small molecular weight, higher stability, excellent programmability, diversity, and low cost have brought them to the forefront of the nucleic acid-based therapeutic arsenal available for cancers. In recent years, considerable efforts have been undertaken to assess a variety of DNAzymes against different cancers. However, their therapeutic application is constrained by the low delivery efficiency, cellular uptake, and target detection within the tumour microenvironment. Thus, there is a pursuit to identify efficient delivery methods in vivo before the full potential of DNAzymes in cancer therapy is realized. In this light, a review of the recent advances in the use of DNAzymes against cancers in preclinical and clinical settings is valuable to understand its potential as effective cancer therapy. We have thus sought to firstly provide a brief overview of construction and recent improvements in the design of DNAzymes. Secondly, this review stipulates the efficacy, safety, and tolerability of DNAzymes developed against major hallmarks of cancers tested in preclinical and clinical settings. Lastly, the recent advances in DNAzyme delivery systems along with the challenges and prospects for the clinical application of DNAzymes as cancer therapy are also discussed.

Constructing a DNAzyme Delivery and Expression System for Escherichia coli: A Prototype for Phage Therapy

Antisense oligonucleotides exhibit high potential for use as therapeutic agents. '10-23' DNAzymes are antisense molecules with a high chemical stability and catalytic efficiency. In the present study, we developed a phagemid containing a DNAzyme expression system regulated by two promoters. One of these promoters, pA1, promotes constitutive expression of Moloney murine leukemia virus reverse transcriptase (MoMuLV-RT). The other promoter, plac, regulates transcription of the RNA substrate from which MoMuLV-RT produces the DNAzyme by reverse transcription. The ftsZ DNAzyme was used to validate this expression system in the phagemid, named pDESCP. ftsZ DNAzyme expression altered the morphological pattern of Escherichia coli from a bacillary to filamentous form. In E. coli FtsZ is the primary component of the cell division apparatus, forming a structure known as Z-ring, which is the place of division. It is suggested that the DNAzyme ftsZ is decreasing the translation of this protein. Delivery of pDESCP into F+ strain of E. coli cells, using VCSM13, and the possible insertion of other DNAzymes into the cassette makes this phagemid an important prototype for phage therapy.

Metal-Dependent DNAzymes for the Quantitative Detection of Metal Ions in Living Cells: Recent Progress, Current Challenges, and Latest Results on FRET Ratiometric Sensors

Many different metal ions are involved in various biological functions including metallomics and trafficking, and yet there are currently effective sensors for only a few metal ions, despite the first report of metal sensors for calcium more than 40 years ago. To expand upon the number of metal ions that can be probed in biological systems, we and other laboratories employ the in vitro selection method to obtain metal-specific DNAzymes with high specificity for a metal ion and then convert these DNAzymes into fluorescent sensors for these metal ions using a catalytic beacon approach. In this Forum Article, we summarize recent progress made in developing these DNAzyme sensors to probe metal ions in living cells and in vivo, including several challenges that we were able to overcome for this application, such as DNAzyme delivery, spatiotemporal control, and signal amplification. Furthermore, we have identified a key remaining challenge for the quantitative detection of metal ions in living cells and present a new design and the results of a Förster resonance energy transfer (FRET)-based DNAzyme sensor for the ratiometric quantification of Zn2+ in HeLa cells. By converting existing DNAzyme sensors into a ratiometric readout without compromising the fundamental catalytic function of the DNAzymes, this FRET-based ratiometric DNAzyme design can readily be applied to other DNAzyme sensors as a major advance in the field to develop much more quantitative metal-ion probes for biological systems.

Versatile Catalytic Deoxyribozyme Vehicles for Multimodal Imaging-Guided Efficient Gene Regulation and Photothermal Therapy.

Catalytic deoxyribozyme has great potential for gene regulation, but the poor efficiency of the cleavage of mRNA and the lack of versatile DNAzyme vehicles remain big challenges for potent gene therapy. By the rational designing of a diverse vehicle of polydopamine-Mn2+ nanoparticles (MnPDA), we demonstrate that MnPDA has integrated functions as an effective DNAzyme delivery vector, a self-generation source of DNAzyme cofactor for catalytic mRNA cleavage, and an inherent therapeutic photothermal agent as well as contrast agent for photoacoustic and magnetic resonance imaging. Specifically, the DNAzyme-MnPDA nanosystem protects catalytic deoxyribozyme from degradation and enhances cellular uptake efficiency. In the presence of intracellular glutathione, the nanoparticles are able to in situ generate free Mn2+ as a cofactor of DNAzyme to effectively trigger the catalytic cleavage of mRNA for gene silencing. In addition, the nanosystem shows high photothermal conversion efficiency and excellent stability against photothermal processing and degradation in complex environments. Unlike previous DNAzyme delivery vehicles, this vehicle exhibits diverse functionalities for potent gene regulation, allowing multimodal imaging-guided synergetic gene regulation and photothermal therapy both in vitro and in vivo.

Therapeutic Potential of DNAzyme Loaded on Chitosan/Cyclodextrin Nanoparticle to Recovery of Chemosensitivity in the MCF-7 Cell Line.

Commonly, acquired resistances to anticancer drug are mediated by overexpression of a membrane-associated protein that encode via multi-drug resistance gene-1 (MDR1). Herein, the mRNA-cleaving DNAzyme that targets the mRNA of MDR1 gene in doxorubicin-resistant breast cancer cell line (MCF-7/DR) loaded on the chitosan β-cyclodextrin complexes was used as a tropical agent. Chitosan/β-cyclodextrin complexes were used to deliver DNAzymes into cancer cells. Determination of the physicochemical characteristics of the particles was done by photon correlation spectroscopy and scanning electron microscopy. The encapsulation efficiency of the complexes was tested by using gel retardation assay. Positively charged nanoparticles interacted with DNAzyme that could perform as an efficient DNAzyme transfection system. The rationale usage of this platform is to sensitize MCF-7/DR to doxorubicin by downregulating the drug-resistance gene MDR1. Results demonstrated a downregulation of MDR1 mRNAs in MCF-7/DR/DNZ by real-time PCR, compared to the MCF-7/DR as control. WST1 assay showed the 22-fold decrease in drug resistance on treated cells 24 h after transfection. Results showed the intracellular accumulation of Rh123 increased in the treated cells with DNAzyme. Results suggested a potential platform in association with chemotherapy drug for cancer therapy and indicated extremely efficient at delivery of DNAzyme in restoring chemosensitivity.

Delivery of DNAzyme targeting aurora kinase A to inhibit the proliferation and migration of human prostate cancer.

Herein, a polyethylenimine derivative N-acetyl-l-leucine-polyethylenimine (N-Ac-l-Leu-PEI) was employed as a carrier to achieve the delivery of DNAzyme targeting aurora kinase A using PC-3 cell as a model. Flow cytometry and confocal laser scanning microscopy demonstrated that the derivative could realize the cellular uptake of nanoparticles in an energy-dependent and clathrin-mediated pathway and obtain a high DNAzyme concentration in the cytoplasm through further endosomal escape. After DNAzyme transfection, expression level of aurora kinase A would be downregulated at the protein level. Meanwhile, the inhibition of cell proliferation was observed through 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and cell colony formation assay, attributing to the activation of apoptosis and cell cycle arrest. Through flow cytometric analysis, an early apoptotic ratio of 25.93% and G2 phase of 22.58% has been detected after N-Ac-l-Leu-PEI-mediated DNAzyme transfection. Finally, wound healing and Transwell migration assay showed that DNAzyme transfection could efficiently inhibit the cell migration. These results demonstrated that N-Ac-l-Leu-PEI could successfully mediate the DNAzyme delivery and downregulate the expression level of aurora kinase A, triggering a significant inhibitory effect of excessive proliferation and migration of tumor cells.

Hydrophobic N-acetyl-l-leucine grafted polyethylenimine as an efficient carrier for DNAzyme delivery

Hydrophobic N-acetyl-l-leucine grafted polyethylenimine as an efficient carrier for DNAzyme delivery

Chimeric nucleolin aptamer with survivin DNAzyme for cancer cell targeted delivery.

A chimeric aptamer-DNAzyme conjugate was generated for the first time using a nucleolin aptamer (NCL-APT) and survivin Dz (Sur_Dz) and exhibited the targeted killing of cancer cells. This proof of concept of using an aptamer for the delivery of DNAzyme can be applied to other cancer types to target survivin in cancer cells in a specific manner.

Functionalization of cationic poly(p-phenylene ethynylene) with dendritic polyethylene enables efficient DNAzyme delivery for imaging Pb2+ in living cells.

We report here an effective Pb2+-dependent DNAzyme (8-17 DNAzyme) delivery system based on the water-soluble dendritic polyethylene-cationic poly(p-phenylene ethynylene) for successfully imaging Pb2+ in living cells. For utilizing the 8-17 DNAzyme and its unique ability to catalyze a phosphodiester bond cleavage reaction in the presence of Pb2+, the distinctive conjugated polymer-based polyvalent nanocarrier design manages to load and transport 8-17 DNAzyme across cell membranes, and to realize the fluorescence imaging of Pb2+ in living cells. As shown by the confocal microscopy and flow cytometry observations, the fluorescence of Cy5.5 is obviously activated under the conditions of incubation with Pb2+, compared with the absence of Pb2+. Taken together, the study demonstrates the combination of the molecular-wire effect with "dendrimer effects" on their effective DNAzyme delivery and their cellular imaging Pb2+.

Selective Inhibition of the Master Regulator Transcription Factor Egr‐1 With Catalytic Oligonucleotides Reduces Myocardial Injury and Improves Left Ventricular Systolic Function in a Preclinical Model of Myocardial Infarction

BackgroundEgr‐1 is implicated in the pathogenesis of myocardial ischemia–reperfusion injury. The aim of this study was to ascertain the effectiveness of intracoronary delivery of DNAzyme targeting the transcription factor Egr‐1 at reperfusion following experimental myocardial ischemia.Methods and ResultsFunctional DNAzyme targeting Egr‐1 or a size‐matched scrambled control were delivered via the intracoronary route immediately on reperfusion after 60 minutes' balloon occlusion of the left anterior descending coronary artery in a pig model of myocardial I/R injury (n=7 per treatment group). Heart function and extent of myocardial infarction were determined following intervention by echocardiography and cardiac magnetic resonance imaging, respectively. Hearts were removed and examined for molecular and histological markers of inflammation and apoptosis. Administration of functional DNAzyme led to an overall decrease in the expression of inflammatory markers including intracellular adhesion molecule‐1, tissue factor, and complement 3, with associated decreases in the extent of neutrophil infiltration, oxidative damage, and subsequent apoptosis within the infarct border zone. Functional significance was indicated by an increase in salvaged left ventricular myocardium (P=0.012), ejection fraction (P=0.002), and fractional area change (P=0.039) in the functional DNAzyme–treated group compared with the control.ConclusionsEgr‐1 silencing through intracoronary delivery of a targeting DNAzyme at the time of reperfusion following acute myocardial ischemia decreases myocardial inflammation and apoptosis leading to improved cardiac function.

DNAzyme Delivery Approaches in Biological Settings

DNAzymes are DNA-based catalytic molecules that have potential use in a range of disorders where the targeted gene plays an important role in disease pathogenesis. DNAzymes are at a comparatively early developmental stage as alternatives to conventional therapies. The biological action of DNAzymes on target mRNA requires efficient delivery into target cells and this hurdle has hampered their broader use, particularly in systemic settings. DNAzymes have been delivered in naked form without a carrier or combined with agents such as polymers and liposomes. This article reviews these and other delivery approaches and offers perspectives on future methodologies for improved DNAzyme delivery and utility as novel drugs.

Functional delivery of DNAzyme with iron oxide nanoparticles for hepatitis C virus gene knockdown

DNAzyme is an attractive therapeutic oligonucleotide which enables cleavage of mRNA in a sequence-specific manner and thus, silencing target gene. A particularly important challenge in achieving the successful down-regulation of gene expression is to efficiently deliver DNAzymes to disease sites and cells. Here, we report the nanoparticle-assisted functional delivery of therapeutic DNAzyme for the treatment of hepatitis C by inducing knockdown of hepatitis C virus (HCV) gene, NS3. HCV NS3 gene encodes helicase and protease which are essential for the virus replication. The nanocomplex showed efficient NS3 knockdown while not evoking undesired immune responses or notable cytotoxicity. We also demonstrated the DNAzyme conjugated nanoparticle system could be applicable in vivo by showing the accumulation of the nanoparticles in liver, and more specifically, in hepatocytes. We believe that the present work is a successful demonstration of effective, functional, non-immunostimulatory DNAzyme delivery system based on inorganic nanoparticles with high potential for further therapeutic application of DNAzyme in the treatment of hepatitis C.

Specific Inhibition of BCR-ABL Gene Expression in Acute Lymphoblastic Leukemia by Catalytic DNAzymes.

The BCR-ABL fusion protein p190 resulting from the translocation t(9;22) exhibits dysregulated tyrosine kinase activity and was shown to cause acute lymphoblastic leukemia (ALL). Detection of the BCR-ABL fusion gene in childhood ALL is associated with an adverse prognosis and defines a group of high risk patients. Because the BCR-ABL gene fusion is specific for leukemic cells it represents an ideal target for leukemia specific treatment approaches.Catalytic DNAzymes are able to cleave mRNA in a sequence specific manner, causing inhibition of protein translation from the DNAzyme targeted mRNA both in vitro and in vivo. In order to cut off the BCR-ABL driven malignant proliferation, we designed DNAzymes to impede the expression of p190 BCR-ABL by cleaving the BCR-ABL mRNA adjacent to the fusion site. One construct was found that cleaved the target mRNA efficiently and specifically leaving BCR and ABL, relevant for normal cell survival and proliferation, unaffected. Activity and specificity of the BCR-ABL DNAzyme was investigated in cleavage assays with in vitro transcribed BCR-ABL, BCR and ABL mRNA. DNAzymes were delivered to cultured BCR-ABL+ ALL cells by lipid transfection. The efficiency of cellular delivery reached 90% as studied by flow cytometry, fluorescence microscopy and confocal microscopy after transfection of FITC labeled DNAzymes. To control for unspecific effects of DNAzyme delivery as well as for antisense effects, a catalytically inactive DNAzyme still exhibiting BCR-ABL antisense activity was designed. Fourty-eight hours after a single treatment of BCR-ABL+ ALL-cells with DNAyzmes the BCR-ABL mRNA concentration, as measured by quantitative real-time RT-PCR, was significantly reduced by 56% and 66% compared to controls treated with the inactivated DNAzyme and to untreated cells, respectively. Western blot analysis showed a decrease in p190 protein levels after DNAzyme treatment in comparison to the control treated with inactive DNAzyme as well as to the untreated cells. Most noteworthy, four days after a single DNAzyme treatment the net growth of BCR-ABL+ ALL cells treated with the active DNAzyme was inhibited by 68% compared to the untreated control.From these data we conclude, firstly, DNAzymes targeting mRNA coding for the minor BCR-ABL variant are able to significantly reduce the amount of fusion mRNA in the cells, leading to a reduction in protein expression, followed by the inhibition of BCR-ABL driven proliferation of ALL cells. Secondly, this exemplified setting gives a hint that DNAzymes might be of therapeutic use in hematopoietic malignancies associated with specific mutations, expressing oncogenic fusion genes or overexpressing oncogenic genes.