Download the Free Unbound MEDLINE PubMed App to your smartphone or tablet.
Available for iPhone, iPad, iPod touch, and Android.
naked DNA [keywords]
- Ultrasensitive and Closed-Tube Colorimetric Loop-Mediated Isothermal Amplification Assay Using Carboxyl-Modified Gold Nanoparticles. [JOURNAL ARTICLE]
- Small 2014 Mar 13.
LAMP ends up in purple/red: The incorporation of 11-mercaptoundecanoic acid-modified gold nanoparticles into loop-mediated isothermal amplification (LAMP) reaction enables as few as 200 copies of a target DNA sequence to be detected by the naked eye. The color is controlled by magnesium ion-templated aggregation, and in the presence of the target, the LAMP reaction by-product pyrophosphate ion leads to particle deaggregation.
- Amplified Single Base-Pair Mismatch Detection via Aggregation of Exonuclease-Sheared Gold Nanoparticles. [JOURNAL ARTICLE]
- Anal Chem 2014 Mar 10.
Single nucleotide polymorphism (SNP) detection is important for early diagnosis, clinical prognostics and disease prevention, and a rapid and sensitive low-cost SNP detection assay would be valuable for resource-limited clinical settings. We present a simple platform that enables sensitive, naked-eye detection of SNPs with minimal reagent and equipment requirements at room temperature within 15 minutes. SNP detection is performed in a single tube with one set of DNA probe-modified gold nanoparticles (AuNPs), a single exonuclease ( Exo III), and the target in question. Exo III's apurinic endonucleolytic activity differentially processes hybrid duplexes between the AuNP-bound probe and DNA targets that are perfectly matched or contain a single-base mismatch. For perfectly matched targets, Exo III's exonuclease activity facilitates a process of target recycling that rapidly shears DNA probes from the particles, generating an AuNP aggregation-induced color change, whereas no such change occurs for mismatched targets. This color change is easily observed with as little as 2 nM target, 100-fold lower than the target concentration required for reliable naked eye observation with unmodified AuNPs in well-optimized reaction conditions. We further demonstrate that this system can effectively discriminate a range of different mismatches.
- G-quadruplex DNAzymes-induced highly selective and sensitive colorimetric sensing of free heme in rat brain. [JOURNAL ARTICLE]
- Analyst 2014 Mar 6.
Direct selective determination of free heme in the cerebral system is of great significance due to the crucial roles of free heme in physiological and pathological processes. In this work, a G-quadruplex DNAzymes-induced highly sensitive and selective colorimetric sensing of free heme in rat brain is established. Initially, the conformation of an 18-base G-rich DNA sequence, PS2.M (5'-GTGGGTAGGGCGGGTTGG-3'), in the presence of K(+), changes from a random coil to a "parallel" G-quadruplex structure, which can bind free heme in the cerebral system with high affinity through π-π stacking. The resulted heme/G-quadruplex complex exhibits high peroxidase-like activity, which can be used to catalyze the oxidation of colorless ABTS(2-) to green ABTS˙(-) by H2O2. The concentration of heme can be evaluated by the naked eye and determined by UV-vis spectroscopy. The signal output showed a linear relationship for heme within the concentration range from 1 to 120 nM with a detection limit of 0.637 nM. The assay demonstrated here was highly selective and free from the interference of physiologically important species such as dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), ascorbate acid (AA), cysteine, uric acid (UA), glucose and lactate in the cerebral system. The basal dialysate level of free heme in the microdialysate from the striatum of adult male Sprague-Dawley rats was determined to be 32.8 ± 19.5 nM (n = 3). The analytic protocol possesses many advantages, including theoretical simplicity, low-cost technical and instrumental demands, and responsible detection of heme in rat brain microdialysate.
- Treatment of phenylketonuria using minicircle-based naked-DNA gene transfer to murine liver. [JOURNAL ARTICLE]
- Hepatology 2014 Feb 28.
Host immune response to viral vectors, persistence of non-integrating vectors and sustained transgene expression are among the major challenges in gene therapy. To overcome these hurdles, we successfully used minicircle (MC) naked-DNA vectors devoid of any viral or bacterial sequences for the long-term treatment of murine phenylketonuria, a model for a genetic liver defect. MC-DNA vectors expressed the murine phenylalanine hydroxylase (Pah) cDNA from a liver-specific promoter coupled to a de novo designed hepatocyte-specific regulatory element, designated as P3, which is a cluster of evolutionary conserved transcription factor binding site. MC-DNA vectors were subsequently delivered to the liver by a single hydrodynamic tail vein (HTV) injection. The MC-DNA vector normalized blood phenylalanine concomitant with reversion of hypopigmentation in a dose dependent manner for more than one year, whereas the corresponding parental plasmid did not result in any phenylalanine clearance. MC vectors persisted in an episomal state in the liver consistent with sustained transgene expression and hepatic PAH enzyme activity without any apparent adverse effects. Moreover, 14-20% of all hepatocytes expressed transgenic PAH, and the expression was observed exclusively in the liver and predominately around pericentral areas of the hepatic lobule while there was no transgene expression in periportal areas. Conclusion: This study demonstrates that MC technology offers an improved safety profile and has the potential for the genetic treatment of liver diseases. (Hepatology 2014;).
- Photosensitive Fluorescent Dye Contributes to Phototoxicity and Inflammatory Responses of Dye-doped Silica NPs in Cells and Mice. [Journal Article]
- Theranostics 2014; 4(4):445-59.
Dye-doped fluorescent silica nanoparticles provide highly intense and photostable fluorescence signals. However, some dopant dye molecules are photosensitive. A widely-used photosensitive fluorescent dopant, RuBpy, was chosen to systematically investigate the phototoxicity of the dye-doped silica nanoparticles (NPs). We investigated cell viability, DNA damage, and Reactive Oxygen Species (ROS) levels in alveolar macrophages using the dye-doped NPs with or without irradiation. Our results showed that the RuBpy-doped silica NPs could induce significant amount of ROS, DNA damage, apoptosis and impaired proliferation in MH-S cells. In vivo studies in mice showed that RuBpy-doped silica NPs induced significant inflammatory cytokine production and lowered expression in signaling proteins such as ERK1/2 and NF-κB as well as increased lung injury determined by myeloperoxidase and lipid peroxidation. Strikingly, we also found that both RuBpy alone and NPs induced systemic signaling activation in the kidney compared to the liver and lung where showed highly selective signaling patterns, which is more pronounced than RuBpy-doped silica NPs. Moreover, we discovered a critical biomarker (e.g., HMGB1) for silica NPs-induced stress and toxicity and demonstrated differentially-regulated response patterns in various organs. Our results indicate for the first time that the RuBpy-doped silica NPs may impose less inflammatory responses but stronger thermotherapeutic effects on target cells in animals than naked NPs in a time- and dose-dependent manner.
- DNA-Gold Nanoparticle Conjugates-Based Nanoplasmonic Probe for Specific Differentiation of Cell Types. [JOURNAL ARTICLE]
- Anal Chem 2014 Mar 4.
The direct analysis of cancerous cells provides a new way for cancer detection that obviates cell lysis and other tedious steps (e.g., enrichment, purification, and amplification steps). However, the analysis of different cell types remains challenging due to the subtle differences in cell surface features. Here, we have demonstrated nanoplasmonic differentiation of cell types by using DNA-gold nanoparticle nanoconjugates (DNA-AuNPs). Our strategy relies on cross reactive receptors (a collection of DNA-AuNPs) that are employed to bind the different cells that produce fingerprint-like patterns for each type of cell. Because of the enhanced nanoplasmonic effect of AuNPs via seeded-growth, we could effectively differentiate various cell lines, e.g., 786-O, L929, Hela, and RAW264.7, with dark-field microscopy or even naked eyes.
- Constitutive and inducible co-expression systems for non-viral osteoinductive gene therapy. [Journal Article, Research Support, Non-U.S. Gov't]
- Eur Cell Mater 2014.:166-84; discussion 184.
Tissue regenerative gene therapy requires expression strategies that deliver therapeutic effective amounts of transgenes. As physiological expression patterns are more complex than high-level expression of a singular therapeutic gene, we aimed at constitutive or inducible co-expression of 2 transgenes simultaneously. Co-expression of human bone morphogenetic protein 2 and 7 (BMP2/7) from constitutively expressing and doxycycline inducible plasmids was evaluated in vitro in C2C12 cells with osteocalcin reporter gene assays and standard assays for osteogenic differentiation. The constitutive systems were additionally tested in an in vivo pilot for ectopic bone formation after repeated naked DNA injection to murine muscle tissue. Inductor controlled differentiation was demonstrated in vitro for inducible co-expression. Both co-expression systems, inducible and constitutive, achieved significantly better osteogenic differentiation than single factor expression. The potency of the constitutive co-expression systems was dependent on relative expression cassette topology. In vivo, ectopic bone formation was demonstrated in 6/13 animals (46% bone formation efficacy) at days 14 and 28 in hind limb muscles as proven by in vivo µCT and histological evaluation. In vitro findings demonstrated that the devised single vector BMP2/7 co-expression strategy mediates superior osteoinduction, can be applied in an inductor controlled fashion and that its efficiency is dependent on expression cassette topology. In vivo results indicatethatco-expression of BMP2/7 applied by non-viral naked DNA gene transfer effectively mediates bone formation without the application of biomaterials, cells or recombinant growth factors, offering a promising alternative to current treatment strategies with potential for clinical translation in the future.
- Strategies and hurdles using DNA vaccines to fish. [Journal Article]
- Vet Res 2014; 45(1):21.
DNA vaccinations against fish viral diseases as IHNV at commercial level in Canada against VHSV at experimental level are both success stories. DNA vaccination strategies against many other viral diseases have, however, not yet yielded sufficient results in terms of protection. There is an obvious need to combat many other viral diseases within aquaculture where inactivated vaccines fail. There are many explanations to why DNA vaccine strategies against other viral diseases fail to induce protective immune responses in fish. These obstacles include: 1) too low immunogenicity of the transgene, 2) too low expression of the transgene that is supposed to induce protection, 3) suboptimal immune responses, and 4) too high degradation rate of the delivered plasmid DNA. There are also uncertainties with regard distribution and degradation of DNA vaccines that may have implications for safety and regulatory requirements that need to be clarified. By combining plasmid DNA with different kind of adjuvants one can increase the immunogenicity of the transgene antigen - and perhaps increase the vaccine efficacy. By using molecular adjuvants with or without in combination with targeting assemblies one may expect different responses compared with naked DNA. This includes targeting of DNA vaccines to antigen presenting cells as a central factor in improving their potencies and efficacies by means of encapsulating the DNA vaccine in certain carriers systems that may increase transgene and MHC expression. This review will focus on DNA vaccine delivery, by the use of biodegradable PLGA particles as vehicles for plasmid DNA mainly in fish.
- Detecting the Nonviable and Heat-Tolerant Bacteria in Activated Sludge by Minimizing DNA from Dead Cells. [JOURNAL ARTICLE]
- Microb Ecol 2014 Feb 18.
Propidium monoazide (PMA) has been used to determine viable microorganisms for clinical and environmental samples since selected naked DNA which was covalently cross-linked by this dye could not be PCR-amplified. In this study, we applied PMA to the activated sludge samples composed of complex bacterial populations to investigate the viability of human fecal bacteria and to determine the heat-tolerant bacteria by high-throughput sequencing of 16S ribosomal DNA (rDNA) V3 region. The methodological evaluation suggested the validity, and about 2-3 magnitude signals decreasing from the stained DNA were observed. However, the nest PCR, which was previously conducted to further minimize signals from dead cells, seemed not suitable perhaps due to the limitation of the primers. On one hand, for typical human fecal bacteria, less than half of them were viable, and most genera exhibited the similar viable percentages. It was interesting that many "unclassified bacteria" showed low viability, implying their sensitivity to environmental change. On the other hand, after heating at 60 °C for 4 h, the bacteria with high survival rate in activated sludge samples included those reported thermophiles or heat-tolerant lineages, such as Anoxybacillus and diverse species in Actinobacteria, and some novel ones, such as Gp16 subdivision in Acidobacteria. In summary, our results took a glance at the fate of fecal bacteria during sewage treatment and established an example for identifying tolerant species to lethal shocks in a complex community.
- Built-in adjuvanticity of genetically and protein-engineered chimeric molecules for targeting of influenza A peptide epitopes. [JOURNAL ARTICLE]
- Immunol Res 2014 Feb 11.
Highly purified, subunit, or synthetic viral antigens are known to be weakly immunogenic and potentate only the antibody, rather than cell-mediated immune responses. An alternative approach for inducing protective immunity with small viral peptides would be the direct targeting of viral epitopes to the immunocompetent cells by DNA vaccines encoding antibody fragments specific to activating cell surface co-receptor molecules. Here, we are exploring as a new genetic vaccine, a DNA chimeric molecule encoding a T and B cell epitope-containing influenza A virus hemagglutinin peptide joined to sequences encoding a single-chain variable fragment antibody fragment specific for the costimulatory B cell complement receptors 1 and 2. This recombinant DNA molecule was inserted into eukaryotic expression vector and used as a naked DNA vaccine in WT and CR1/2 KO mice. The intramuscular administration of the DNA construct resulted in the in vivo expression of an immunogenic chimeric protein, which cross-links cell surface receptors on influenza-specific B cells. The DNA vaccination was followed by prime-boosting with the protein-engineered replica of the DNA construct, thus delivering an activation intracellular signal. Immunization with an expression vector containing the described construct and boosting with the protein chimera induced a strong anti-influenza cytotoxic response, modulation of cytokine profile, and a weak antibody response in Balb/c mice. The same immunization scheme did not result in generation of influenza-specific response in mice lacking the target receptor, underlining the molecular adjuvant effect of receptor targeting.