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naked DNA [keywords]
- The impact of exogenous DNA on the structure of sperm of olive flounder (Paralichthys olivaceus). [JOURNAL ARTICLE]
- Anim Reprod Sci 2014 Jul 3.
Sperm-mediated gene transfer (SMGT) is a promising transgenic technology that relies on the capability of sperm to internalize exogenous DNA. In marine fish, however, the interaction between sperm and exogenous DNA appears to be deficient. Here, we demonstrated significant DNase activity in the seminal plasma of the olive flounder. When incubated with naked-DNA, the spermatozoa lost their structural integrity, including the head, mitochondria and flagellum, in an incubation time-dependent manner. However, internalization of a liposome-DNA complex resulted in the structural integrity of the spermatozoa being maintained, even when using incubation times of up to 50min. We concluded that in the olive flounder, SMGT is possible by integrating liposome-DNA complexes, rather than naked-DNA alone, into the sperm. In brief, removal of the seminal plasma and packaging the exogenous DNA were necessary for successful SMGT in the olive flounder.
- Cervical Cancer Gene Therapy by Gene Loaded PEG-PLA Nanomedicine. [Journal Article]
- Asian Pac J Cancer Prev 2014; 15(12):4915-8.
Background and Aims:Advances in the treatment of cervical cancer over the last decade have predominantly involved the development of genes directed at molecular targets. Gene therapy is recognized to be a novel method for the treatment of cervical cancer. Genes can be administered into target cells via nanocarriers. This study aimed to develop systemically administrable nano-vectors. Floate (Fa) containing gene loaded nanoparticles (NPs) could target HeLa human cervical cancer cells through combination with receptors on the cells to increase the nuclear uptake of genetic materials.
Methods:Fa was linked onto Poly (ethylene glycol)-b-poly (D, L-lactide) (PEG-PLA) to form Fa-PEG-PLA, and the resulting material was used to load plasmids of enhanced green fluorescence protein (pEGFP) to obtain gene loaded nanoparticles (Fa-NPs/DNA). Physical-chemical characteristics, in vitro release and cytotoxicity of Fa-NPs/DNA were evaluated. The in vitro transfection efficiency of Fa-NPs/ DNA was evaluated in HeLa cells and human umbilical vein endothelial cells (HUVEC). PEG-PLA without Fa was used to load pEGFP from NPs/DNA as a control.
Results:Fa-NPs/DNA has a particle size of 183 nm and a gene loading quantity of 92%. After 72h of transfection, Fa-NPs/DNA displayed over 20% higher transfection efficiency than NPs/DNA and 40% higher than naked DNA in HeLa cells. However, in HUVECs, no significant difference appeared between Fa-NPs/DNA and NPs/DNA.
Conclusions:Fa-PEG-PLA NPs could function as excellent materials for gene loading. This nano-approach could be used as tumor cell targeted medicine for the treatment of cervical cancer.
- Cationic Surfactant-Based Colorimetric Detection of Plasmodium Lactate Dehydrogenase, a Biomarker for Malaria, Using the Specific DNA Aptamer. [JOURNAL ARTICLE]
- PLoS One 2014; 9(7):e100847.
A simple, sensitive, and selective colorimetric biosensor for the detection of the malarial biomarkers Plasmodium vivax lactate dehydrogenase (PvLDH) and Plasmodium falciparum LDH (PfLDH) was demonstrated using the pL1 aptamer as the recognition element and gold nanoparticles (AuNPs) as probes. The proposed method is based on the aggregation of AuNPs using hexadecyltrimethylammonium bromide (CTAB). The AuNPs exhibited a sensitive color change from red to blue, which could be seen directly with the naked eye and was monitored using UV-visible absorption spectroscopy and transmission electron microscopy (TEM). The reaction conditions were optimized to obtain the maximum color intensity. PvLDH and PfLDH were discernible with a detection limit of 1.25 pM and 2.94 pM, respectively. The applicability of the proposed biosensor was also examined in commercially available human serum.
- In Vitro-Reconstituted Nucleoids Can Block Mitochondrial DNA Replication and Transcription. [JOURNAL ARTICLE]
- Cell Rep 2014 Jun 25.
The mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000-10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A (TFAM). Here, we reconstitute nucleoid-like particles in vitro and demonstrate that small changes in TFAM levels dramatically impact the fraction of DNA molecules available for transcription and DNA replication. Compaction by TFAM is highly cooperative, and at physiological ratios of TFAM to DNA, there are large variations in compaction, from fully compacted nucleoids to naked DNA. In compacted nucleoids, TFAM forms stable protein filaments on DNA that block melting and prevent progression of the replication and transcription machineries. Based on our observations, we suggest that small variations in the TFAM-to-mtDNA ratio may be used to regulate mitochondrial gene transcription and DNA replication.
- Ultrasound-mediated gene delivery of naked plasmid DNA in skeletal muscles: a case for bolus injections. [REVIEW]
- J Control Release 2014 Jun 27.
Localized gene delivery has many potential clinical applications. However, the nucleic acids (e.g. pDNA and siRNA) are incapable of passively crossing the endothelium, cell membranes and other biological barriers which must be crossed to reach their intracellular targets. A possible solution is the use of ultrasound to burst circulating microbubbles inducing transient permeabilization of surrounding tissues which mediates nucleic acid extravasation and cellular uptake. In this study we report on an optimization of the ultrasound gene delivery technique. Naked pDNA (200μg) encoding luciferase and SonoVue® microbubbles were co-injected intravenously in mice. The hindlimbs skeletal muscles were exposed to ultrasound from a non-focused transducer (1MHz, 1.25MPa, PRI 30s) and injection protocols and total amounts as well as ultrasound parameters were systemically varied. Gene expression was quantified relative to a control using a bioluminescence camera system at day 7 after sonication. Bioluminescence ratios in sonicated / control muscles of up to 101x were obtained. In conclusion, we were able to specifically deliver genetic material to the selected skeletal muscles and overall, the use of bolus injections and high microbubble numbers resulted in increased gene expression reflected by stronger bioluminescence signals. Based on our data, bolus injections seem to be required in order to achieve transient highly concentrated levels of nucleic acids and microbubbles at the tissue of interest which upon ultrasound exposure should lead to increased levels of gene delivery. Thus, ultrasound mediated gene delivery is a promising technique for the clinical translation of localized drug delivery.
- Intelligent layered nanoflare: "lab-on-a-nanoparticle" for multiple DNA logic gate operations and efficient intracellular delivery. [JOURNAL ARTICLE]
- Nanoscale 2014 Jun 26.
DNA strand displacement cascades have been engineered to construct various fascinating DNA circuits. However, biological applications are limited by the insufficient cellular internalization of naked DNA structures, as well as the separated multicomponent feature. In this work, these problems are addressed by the development of a novel DNA nanodevice, termed intelligent layered nanoflare, which integrates DNA computing at the nanoscale, via the self-assembly of DNA flares on a single gold nanoparticle. As a "lab-on-a-nanoparticle", the intelligent layered nanoflare could be engineered to perform a variety of Boolean logic gate operations, including three basic logic gates, one three-input AND gate, and two complex logic operations, in a digital non-leaky way. In addition, the layered nanoflare can serve as a programmable strategy to sequentially tune the size of nanoparticles, as well as a new fingerprint spectrum technique for intelligent multiplex biosensing. More importantly, the nanoflare developed here can also act as a single entity for intracellular DNA logic gate delivery, without the need of commercial transfection agents or other auxiliary carriers. By incorporating DNA circuits on nanoparticles, the presented layered nanoflare will broaden the applications of DNA circuits in biological systems, and facilitate the development of DNA nanotechnology.
- A universal lateral flow biosensor for proteins and DNAs based on the conformational change of hairpin oligonucleotide and its use for logic gate operations. [JOURNAL ARTICLE]
- Biosens Bioelectron 2014 Jun 11.:598-604.
A universal lateral flow biosensor for proteins and DNAs was designed on the base of target-induced conformational changes of hairpin oligonucleotide (HO). CEA (carcinoembryonic antigen) protein and c-DNA were detected both with the naked eye and a strip reader. The scheme of detecting proteins and DNAs were based on the unique molecular recognition properties of HO to the targets to form different quantities of "active" biotin groups on the surface of gold nanoparticles (AuNPs). The output of the strip is the color of the test line, which inspired us to combine strip biosensor with logic gate. Two strip logic gates ("OR" and "INH") were designed in our paper and the combinatorial logic gates in our paper could be used to make high-throughput judgment about what targets were present in the input samples according to the output results. The biosensor facilitates a portable analysis at ambient temperature as it is simple to be conducted and no requirement of training is needed. The strip logic system is proved an excellent selection and can operate effectively as well as in human serum samples. Therefore, we indicate that such logic strips a foreseeable promise in application of intelligent point-of-care and in-field diagnostics.
- Sensitive colorimetric biosensing for methylation analysis of p16/CDKN2 promoter with hyperbranched rolling circle amplification. [JOURNAL ARTICLE]
- Biosens Bioelectron 2014 Jun 10.:593-597.
A simple, fast and sensitive colorimetric biosensing method for DNA methylation analysis was developed by combining methylation-sensitive endonuclease based digestion with hyperbranched rolling circle amplification (HRCA) induced signal enhancement. The assay was carried out on a DNA capture probe modified 96-cell microplate with four sequential steps of target recognition, endonuclease-based digestion, isothermal HRCA, and enzyme-catalyzed colorimetric readout within 3h. The semi-quantitative and precise analysis of methylated DNA could be easily achieved by naked eye and absorbance measurements, respectively. The strategy exhibited excellent detection specificity and accuracy with a log-linear response to methylated DNA from 100fM to 10nM. As a proof of concept, the assay was applied to investigate the methylation status of p16/CDKN2 promoter of breast cancer patients. The methylated p16 concentration was not significantly associated with the clinical parameters. The proposed method allowed efficient methylation detection with simplicity, rapidness, low cost and high sensitivity, showing great promise for application in early diagnosis of methylation-related diseases.
- Cell Transfection with a β-Cyclodextrin-PEI-Propane-1,2,3-Triol Nanopolymer. [JOURNAL ARTICLE]
- PLoS One 2014; 9(6):e100258.
Successful gene therapy necessitates safe and efficient gene transfer. This article describes the use of a cationic polymer, which was synthesized by cross-linking low molecular weight branched poly(ethylenimine) (PEI) with both β-cyclodextrin and propane-1,2,3-triol, for efficient and safe non-viral gene delivery. Experimentation demonstrated that the polymer had a pH buffering capacity and DNA condensing ability comparable to those of PEI 25 kDa. In B16-F0 cells, the polymer increased the transfection efficiency of naked DNA by 700-fold and yielded better transfection efficiencies than Fugene HD (threefold higher) and PEI 25 kDa (fivefold higher). The high transfection efficiency of the polymer was not affected by the presence of serum during transfection. In addition to B16-F0 cells, the polymer enabled efficient transfection of HepG2 and U87 cells with low cytotoxicity. Our results indicated that our polymer is a safe and efficient transfection reagent that warrants further development for in vitro, in vivo and clinical applications.
- Nucleosomal regulation of chromatin composition and nuclear assembly revealed by histone depletion. [JOURNAL ARTICLE]
- Nat Struct Mol Biol 2014 Jun 22.
Nucleosomes are the fundamental unit of chromatin, but analysis of transcription-independent nucleosome functions has been complicated by the gene-expression changes resulting from histone manipulation. Here we solve this dilemma by developing Xenopus laevis egg extracts deficient for nucleosome formation and by analyzing the proteomic landscape and behavior of nucleosomal chromatin and nucleosome-free DNA. We show that although nucleosome-free DNA can recruit nuclear-envelope membranes, nucleosomes are required for spindle assembly and for formation of the lamina and of nuclear pore complexes (NPCs). We show that, in addition to the Ran G-nucleotide exchange factor RCC1, ELYS, the initiator of NPC formation, fails to associate with naked DNA but directly binds histone H2A-H2B dimers and nucleosomes. Tethering ELYS and RCC1 to DNA bypasses the requirement for nucleosomes in NPC formation in a synergistic manner. Thus, the minimal essential function of nucleosomes in NPC formation is to recruit RCC1 and ELYS.