DNAzyme-functionalized porous carbon nanospheres serve as a fluorescent nanoprobe for imaging detection of microRNA-21 and zinc ion in living cells.

The present study shows that a dual-signal nanoprobe consisting of DNAzyme-functionalized porous carbon nanospheres (PCNs) responds to microRNA-21 and zinc ion (Zn2+). The fluorescent probe undergoes an increase in the fluorescence intensity of fluorescein isothiocyanate (FITC) (with excitation/emission wavelengths at 488/517 nm) and the fluorescence intensity of cyanine-5 (Cy5) (with excitation/emission wavelengths at 633/670 nm) in the presence of microRNA-21 and Zn2+. The recognition between microRNA-21 and its complementary strand in the PCNs induces the separation of Zn2+-specific DNAzyme from PCNs, thus resulting in the increase of green fluorescence, and the exogenous Zn2+ triggers the rupture of cleavage strand of DNAzyme and recovery of red fluorescence. This nanoprobe allows us to acquire in vitro the determination of microRNA-21 in the range of 2-300 nM with a detection limit of 0.57 nM and the determination of Zn2+ in the range 2-100 nM with a detection limit of 0.43 nM, and in situ simultaneous imaging in MCF-7 breast cancer cells. Therefore, this strategy permits to obtain the expression levels of different biomarkers in living cells, providing a useful tool for diagnosis of cancers and understanding their biological process. Graphical abstract Schematic representation of the DNAzyme-functionalized porous carbon nanospheres for the imaging analysis of microRNA-21 and Zn2+ in living cells.

Optical Control of Metal Ion Probes in Cells and Zebrafish Using Highly Selective DNAzymes Conjugated to Upconversion Nanoparticles.

Spatial and temporal distributions of metal ions in vitro and in vivo are crucial in our understanding of the roles of metal ions in biological systems, and yet there is a very limited number of methods to probe metal ions with high space and time resolution, especially in vivo. To overcome this limitation, we report a Zn2+-specific near-infrared (NIR) DNAzyme nanoprobe for real-time metal ion tracking with spatiotemporal control in early embryos and larvae of zebrafish. By conjugating photocaged DNAzymes onto lanthanide-doped upconversion nanoparticles (UCNPs), we have achieved upconversion of a deep tissue penetrating NIR 980 nm light into 365 nm emission. The UV photon then efficiently photodecages a substrate strand containing a nitrobenzyl group at the 2'-OH of adenosine ribonucleotide, allowing enzymatic cleavage by a complementary DNA strand containing a Zn2+-selective DNAzyme. The product containing a visible FAM fluorophore that is initially quenched by BHQ1 and Dabcyl quenchers is released after cleavage, resulting in higher fluorescent signals. The DNAzyme-UCNP probe enables Zn2+ sensing by exciting in the NIR biological imaging window in both living cells and zebrafish embryos and detecting in the visible region. In this study, we introduce a platform that can be used to understand the Zn2+ distribution with spatiotemporal control, thereby giving insights into the dynamical Zn2+ ion distribution in intracellular and in vivo models.

Two-Photon DNAzyme-Gold Nanoparticle Probe for Imaging Intracellular Metal Ions.

RNA-cleaving DNAzymes have been demonstrated as a promising platform for sensing metal ions. However, the poor biological imaging performance of RNA-cleaving DNAzyme-based fluorescent probes has limited their intracellular applications. Compared with traditional one-photon fluorescence imaging, two-photon (TP) fluorescent probes have shown advantages such as increased penetration depth, lower tissue autofluorescence, and reduced photodamage. Herein, for the first time, we developed an RNA-cleaving DNAzyme-based TP imaging probe (TP-8-17ES-AuNP) for Zn2+ detection in living cells by modifying a Zn2+-specific DNAzyme (8-17) with a TP fluorophore (TP-8-17ES) and using gold nanoparticles (AuNPs) for intracellular delivery. The modified TP-8-17ES exhibits good two-photon properties and excellent photostability. For the TP-8-17ES-AuNP, in the absence of Zn2+, the TP fluorophore is quenched by both AuNPs and the molecular quencher. Only in the presence of Zn2+ does the DNAzyme cleave the TP fluorophore-labeled substrate strand, resulting in fluorescence enhancement and TP imaging. Such probe shows remarkable selectivity of Zn2+ over other metal ions existing in the biological environment. Benefiting from the labeled TP fluorophore, the near-infrared (NIR) excited probe has the capability of TP imaging of Zn2+ in living cells and tissue with a deep tissue penetration up to 160 µm. This method can be generally applied to detect other metal ions in biological systems under TP imaging with higher tissue penetration ability and lower phototoxicity.

Antiangiogenic and antitumoral effects mediated by a vascular endothelial growth factor receptor 1 (VEGFR-1)-targeted DNAzyme.

Antiangiogenesis is a promising antitumor strategy that inhibits tumor vascular formation to suppress tumor growth. DNAzymes are synthetic single-strand deoxyribonucleic acid (DNA) molecules that can cleave ribonucleic acids (RNAs). Here, we conducted a comprehensive in vitro selection of active DNAzymes for their activity to cleave the vascular endothelial growth factor receptor (VEGFR-1) mRNA and screened for their biological activity in a matrigel tube-formation assay. Among the selected DNAzymes, DT18 was defined as a lead molecule that was further investigated in several model systems. In a rat corneal vascularization model, DT18 demonstrated significant and specific antiangiogenic activity, as evidenced by the reduced area and vessel number in VEGF-induced corneal angiogenesis. In a mouse melanoma model, DT18 was shown to inhibit B16 tumor growth, whereas it did not affect B16 cell proliferation. We further assessed the DT18 effect in mice with established human nasopharyngeal carcinoma (NPC). A significant inhibition of tumor growth was observed, which accompanied downregulation of VEGFR-1 expression in NPC tumor tissues. To evaluate DT18 effect on vasculature, we performed dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) on the human NPC xenograft mice treated with DT18 and showed a reduction of the parameter of K(trans) (volume constant for transfer of contrast agent), which reflects the condition of tumor microvascular permeability. When examining the safety and tolerability of DT18, intravenous administration of Dz18 to healthy mice caused no substantial toxicities, as shown by parameters such as body weight, liver/kidney function, and histological and biochemical analyses. Taken together, our data suggest that the anti-VEGFR-1 DNAzyme may be used as a therapeutic agent for the treatment of cancer, such as NPC.

The distribution, clearance, and safety of an anti-MMP-9 DNAzyme in normal and MMTV-PyMT transgenic mice.

Catalytic oligonucleotides, known as DNAzymes, are a new class of nucleic acid-based gene therapy that have recently been used in preclinical animal studies to treat various cancers. In this study the systemic distribution, pharmacokinetics, and safety of intravenously administered anti-MMP (matrix metalloproteinase)-9 DNAzyme (AM9D) were determined in healthy FVB and in MMTV-polyoma virus middle T (PyMT) transgenic mice bearing mammary tumors. MMP-9 is known to be involved in tumor cell development, angiogenesis, invasion, and metastasis. Sulfur-35 ((35)S) labeled ([(35)S]-AM9D) administered intravenously, without the use of carrier molecules, to healthy and mammary tumor bearing MMTV-PyMT transgenic mice distributed to all major organs. The order of percentages of [(35)S]-AM9D accumulation in different organs of healthy and MMTV-PyMT mice were blood>liver>kidney>lung>spleen>heart and mammary tumor>blood≈liver>kidney>spleen>lung>heart, respectively. The amount of AM9D accumulated in mammary tumors 2 hours post injection was 0.6% and 0.2% higher than in either blood or liver, respectively, and its rate of initial clearance from mammary tissue was at least 50% slower than the other organs. Approximately 43% of the delivered dosage of [(35)S]-AM9D was cleared from the system via feces and urine over a period of 72 hours. No evidence of acute or chronic cytotoxicity, local or widespread, associated with AM9D treatment (up to 75 mg AM9D /kg of body weight) was observed in the organs examined. These data suggest that DNAzyme in general and AM9D in particular can be used systemically as a therapeutic agent to treat patients with breast cancer or other metastatic and surgically inaccessible tumors.

Toxicity profile of the GATA-3-specific DNAzyme hgd40 after inhalation exposure.

DNAzymes are single-stranded catalytic DNA molecules that bind and cleave specific sequences in a target mRNA molecule. Their potential as novel therapeutic agents has been demonstrated in a variety of disease models. However, no studies have yet addressed their toxicology and safety pharmacology profiles in detail. Here we describe a detailed toxicological analysis of inhaled hgd40, a GATA-3-specific DNAzyme designed for the treatment of allergic bronchial asthma. Subacute toxicity, immunotoxicity, and respiratory, cardiovascular, and CNS safety pharmacology were analyzed in rodents and non-rodents, and genotoxicity was assessed in human peripheral blood. Overall, hgd40 was very well tolerated when delivered by aerosol inhalation or slow intravenous infusion. Only marginal reversible histopathological changes were observed in the lungs of rats receiving the highest dose of inhaled hgd40. The changes consisted of slight mononuclear cell infiltration and alveolar histiocytosis, and moderate hyperplasia of bronchus-associated lymphoid tissue. No local or systemic adverse effects were observed in dogs. No compound-related respiratory, cardiovascular, or CNS adverse events were observed. The only relevant immunological findings were very slight dose-dependent changes in interleukin-10 and interferon-γ levels in bronchoalveolar lavage fluid. Taken together, these results support direct delivery of a DNAzyme via inhalation for the treatment of respiratory disease.

Cytotoxic G-rich oligodeoxynucleotides: putative protein targets and required sequence motif.

It has recently been shown that certain oligodeoxynucleotides (ODNs) designed as catalytic DNA molecules (DNAzymes) exhibit potent cytotoxicity independent of RNA-cleavage activity in a number of cell lines. These cytotoxic ODNs all featured a 5' G-rich sequence and induced cell death by a TLR9-independent mechanism. In this study, we examined the sequence and length dependence of ODNs for cytotoxicity. A G-rich sequence at the 5' terminus of the molecule was necessary for cytotoxicity and the potency of ODNs with active 5' sequences was length dependent. Cytotoxicity appeared to be generally independent of 3' sequence composition, although 3' sequences totally lacking G-nucleotides were mostly inactive. Nucleolin, elongation factor 1-alpha (eEF1A) and vimentin were identified as binding to a cytotoxic ODN (Dz13) using protein pull-down assays and LC-MS/MS. Although these proteins have previously been described to bind G-rich ODNs, the binding of eEF1A correlated with cytotoxicity, whereas binding of nucleolin and vimentin did not. Quiescent non-proliferating cells were resistant to cytotoxicity, indicating cytotoxicity may be cell cycle dependent. Although the exact mechanism of cytotoxicity remains unknown, marked potency of the longer (> or =25 nt) ODNs in particular, indicates the potential of these molecules for treatment of diseases associated with abnormal cell proliferation.