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naked DNA [keywords]
- Enhanced immune response against HIV-1 induced by a heterologous DNA prime-adenovirus boost vaccination using mannosylated polyethyleneimine as DNA vaccine adjuvant. [JOURNAL ARTICLE]
- Int J Nanomedicine 2013.:1843-1854.
The heterologous deoxyribonucleic acid (DNA) prime-adenovirus (AdV) boost vaccination approach has been widely applied as a promising strategy against human immunodeficiency virus (HIV)-1. However, the problem of inefficient delivery and lack of specificity of DNA vaccine remains a major issue. In this paper, to improve the transfection of DNA vaccine and realize dendritic cell targeting, we used mannosylated polyethyleneimine (man-PEI) as a DNA vector carrier.The DNA plasmid encoding antigen HIV gag fragment was constructed by polymerase chain reaction. Then the DNA plasmid was complexed with man-PEI. The in vitro transfection efficiency of man-PEI/DNA was analyzed on DC 2.4 cells. Mice were primed with 25 μg pVAX1-HIV gag plasmid complexed with man-PEI, 100 μg naked pVAX1-HIV gag plasmid, or empty pVAX1 vector and boosted by AdV encoding the same antigen. The antibody titer, CD4(+) and CD8(+) T-cell response, as well as interferon-γ and interleukin-4 levels in serum and in splenocytes culture were analyzed using flow cytometry or enzyme-linked immunosorbent assay to evaluate the immune response. To test a long-term effect of the vaccination regimen, CD8(+) memory T-cell was also detected by flow cytometry.The pVAX1-HIV gag was constructed successfully. The in vitro transfection efficiency in dendritic cells was significantly higher than naked DNA plasmid. Compared with 100 μg naked DNA/AdV group, the immunoglobulin G2a antibody titer, T-cell response percentage, and cytokine production level induced by man-PEI/DNA/AdV group were significantly higher at a lower DNA dose. Also, the man-PEI/DNA could stimulate a memory CD8(+) T-cell response.Owing to the adjuvant effect of man-PEI, the man-PEI/pVAX1-HIV gag priming plus AdV boosting strategy proved to be a potent vaccine candidate against HIV, which could induce a stronger immune response with a lower DNA dose.
- A visible multi-digit DNA keypad lock based on split G-quadruplex DNAzyme and silver microspheres. [Journal Article]
- Chem Commun (Camb) 2013 Jun 18; 49(48):5459-61.
A novel visible multi-digit DNA keypad lock system was fabricated based on split G-quadruplex DNAzyme and silver microspheres. The final result of the keypad lock can be easily recognized by the naked eye and the number of inputs for the keypad lock can be flexibly adjusted. This molecular platform showed excellent scalability and flexibility.
- Size effect of complexed plasmid DNA to gene transfection efficiency of microbubble-mediated sonoporation. [Journal Article]
- J Acoust Soc Am 2013 May; 133(5):3495.
Ultrasound-mediated gene transfection in the presence of microbubbles is a recently developed promising nonviral gene delivery method. Detailed dynamics of pore opening on the cell surface has not been clarified. Especially, the pore size is one of the most essential parameters. In this study, we investigated the size effect of the complexed plasmid DNA (pDNA) on the transfection efficiency by packaging within the polyplex micelles. Both naked pDNA and complexed pDNA were transfected into cultured NIH3T3 cells using ultrasound in the presence of microbubble contrast agent, Sonazoid. The both size of the hydrodynamic diameter of naked and complexed pDNA estimated by a dynamic light scattering measurement were 600 and 120 nm, respectively. The transfection rates of the complexed pDNA evaluated by counting the number of cells that exhibited green fluorescent was 1.67%, while that of the naked pDNA was 0.92%. This efficiency enhancement depending on the size reduction showed that the pore sizes were distributed in the range of pDNA diameters. Since complexation changes the structure of pDNA in size and stability, more detailed study will be discussed in the presentation.
- A colorimetric method for H1N1 DNA detection using rolling circle amplification. [Journal Article]
- Analyst 2013 Jun 21; 138(12):3457-62.
A highly sensitive and specific colorimetry-based rolling circle amplification (RCA) assay has been successfully developed as a method for the effective detection of H1N1 DNA. Specific oligonucleotide and reporter primer probes were designed together with a circular template, and the oligonucleotide probes were attached to the surfaces of magnetic beads (MBs) to form functional MB-DNA conjugates as capture probes for the target H1N1 DNA molecules. Together with the addition of DNA targets and reporter primer probes to the MB-DNA conjugates, sandwiched hybrids were formed. The initiation of RCA amplification using the circular template in the presence of phi29 polymerase allowed for the amplification of a large number of repeat sequences of the single-stranded (ss)-DNA product. This RCA product accumulated gold nanoparticles (AuNPs), resulting in a colorimetric change that could be viewed by the naked eye or detected using UV-vis spectroscopy. According to this method, H1N1 DNA could be detected at the 1 pmol L(-1) level. This platform exhibited design convenience, simplicity, and cost-effectiveness, and could be used to provide a new diagnostic assay for H1N1, and other infectious diseases.
- Naked-eye detection of nucleic acids through rolling circle amplification and magnetic particle mediated aggregation. [Journal Article]
- Biosens Bioelectron 2013 Sep 15.:515-9.
We are presenting a new method for sequence-specific and naked-eye detection of nucleic acids (NAs) through isothermal amplification and magnetic particle mediated aggregation. A padlock probe was designed to hybridize to specific sequence on target genes followed by ligation and rolling circle amplification (RCA). Magnetic particles (MPs) were then added into RCA products solution. MPs would physically combine with long linear DNA coils (such as products of RCA reaction) and form visible intertwined aggregates, while no aggregate was observed in the absence of high molecular weight NAs (>1kb). As little as 0.62amol (124fM) of target DNA molecules was detected by the naked eye. It was further applied to detect human papillomavirus type 18 gene in genomic DNA isolated from HeLa cells. This assay is sensitive and low-cost with minimal instrumentation, revealing great potential for molecular diagnostics in developing countries and regions with limited settings.
- The SMC1-SMC3 cohesin heterodimer structures DNA through supercoiling-dependent loop formation. [JOURNAL ARTICLE]
- Nucleic Acids Res 2013 Apr 24.
Cohesin plays a critical role in sister chromatid cohesion, double-stranded DNA break repair and regulation of gene expression. However, the mechanism of how cohesin directly interacts with DNA remains unclear. We report single-molecule experiments analyzing the interaction of the budding yeast cohesin Structural Maintenance of Chromosome (SMC)1-SMC3 heterodimer with naked double-helix DNA. The cohesin heterodimer is able to compact DNA molecules against applied forces of 0.45 pN, via a series of extension steps of a well-defined size ≈130 nm. This reaction does not require ATP, but is dependent on DNA supercoiling: DNA with positive torsional stress is compacted more quickly than negatively supercoiled or nicked DNAs. Un-nicked torsionally relaxed DNA is a poor substrate for the compaction reaction. Experiments with mutant proteins indicate that the dimerization hinge region is crucial to the folding reaction. We conclude that the SMC1-SMC3 heterodimer is able to restructure the DNA double helix into a series of loops, with a preference for positive writhe.
- A Decoy Oligonucleotide to NF-κB Delivered Through Inhalable Particles Prevents LPS-Induced Rat Airway Inflammation. [JOURNAL ARTICLE]
- Am J Respir Cell Mol Biol 2013 Apr 3.
The inflammatory process plays a crucial role in the onset/progression of several lung pathologies including cystic fibrosis (CF), and the involvement of nuclear factor-κB (NF-κB ) is widely recognized. The specific inhibition of NF-κB by decoy oligonucleotides delivered in the lung may be beneficial although rationally designed systems are needed to optimize their pharmacological response. Prompted by this, here we have developed and tested in vivo an inhalable dry powder for prolonged delivery of a decoy oligodeoxynucleotide to NF-κB (dec-ODN), consisting of large porous particles (LPP) based on poly(lactic-co-glycolic) acid (PLGA). First, LPP containing dec-ODN (dec-ODN LPP) have been engineered to meet aerodynamic criteria crucial for pulmonary delivery, to gain an effective loading of dec-ODN, to sustain its release and to preserve its structural integrity in lung lining fluids. Then, we have investigated the effects of dec-ODN LPP in a rat model of lung inflammation induced by intra-tracheal aerosolization of lipopolysaccharide (LPS) from P. aeruginosa. The results show that a single intra-tracheal insufflation of dec-ODN LPP reduced bronchoalveolar neutrophil infiltration induced by LPS up to 72 hours, whereas naked dec-ODN was able to inhibit it only at 6 hours. The persistent inhibition of neutrophil infiltrate was associated to a reduced NF-κB/DNA binding activity as well as interleukin-6 (IL-6), interleukin-8 (IL-8) and mucin-2 (MUC2) mRNA expression in lung homogenates. It is worthy of note that the developed LPP, preventing the accumulation of neutrophils and NF-κB-related gene expression, may provide a new therapeutic option for local treatment of inflammation associated with lung diseases.
- Optimization of The Electroporation Conditions for Transfection of Human Factor IX into The Goat Fetal Fibroblasts. [Journal Article]
- Cell J 2013; 14(4):270-5.
Electroporation is the most common method used for the transfection of DNA. Although this method has been attributed for various cell using different buffer compositions, the effects of DNA concentration on the transfection efficiency has not yet been studied. Here the correlation between the efficiency of electroporation reaction and increments of DNA concentration has been investigated. Following this investigation, a study was set out to produce a transgenic goat which is capable of secreting recombinant human coagulation factor IX in its milk.In this experimental study, a linearized recombinant vector (pBC1) entailing human coagulation factor IX (5.7 kb) cDNA was introduced into goat fetal fibroblast cells (three sub passages) via electroporation in four separate experiments (while other variables were kept constant), beginning with 10 µg DNA per pulse in the first experiment and increments of 10 µg/pulse for the next three reactions. Thereafter, polymerase chain reaction (PCR)-positive cell culture plates were diluted by several factors for further analysis of the transfected wells. Subsequently, positive cells were isolated for fluorescence in situ hybridization. Logistic regression model was used for data analyzing. Significance was defined as p< 0.05.The results showed no significant difference among three first concentrations except for group 1 (10 µg/pulse) and group 3 (30 µg/pulse), but there was a significant difference between these groups and the fourth group (p<0.05), as this group (40 µg/pulse) statistically showed the highest rate of transfection. As the fluorescence in situ hybridization (FISH) results indicated the transgene was integrated in a single position in PCR positive cells.Increasing amount of using the vector 40µg/pulse efficiently increased the number of transfected cells. Besides of voltage and buffer strength which had been previously shown to play a critical role in electroporation efficiency, our results showed an increase in DNA concentration could affect an exponential surge in the electroporation efficiency.
- Probing DNA shape and methylation state on a genomic scale with DNase I. [Journal Article]
- Proc Natl Acad Sci U S A 2013 Apr 16; 110(16):6376-81.
DNA binding proteins find their cognate sequences within genomic DNA through recognition of specific chemical and structural features. Here we demonstrate that high-resolution DNase I cleavage profiles can provide detailed information about the shape and chemical modification status of genomic DNA. Analyzing millions of DNA backbone hydrolysis events on naked genomic DNA, we show that the intrinsic rate of cleavage by DNase I closely tracks the width of the minor groove. Integration of these DNase I cleavage data with bisulfite sequencing data for the same cell type's genome reveals that cleavage directly adjacent to cytosine-phosphate-guanine (CpG) dinucleotides is enhanced at least eightfold by cytosine methylation. This phenomenon we show to be attributable to methylation-induced narrowing of the minor groove. Furthermore, we demonstrate that it enables simultaneous mapping of DNase I hypersensitivity and regional DNA methylation levels using dense in vivo cleavage data. Taken together, our results suggest a general mechanism by which CpG methylation can modulate protein-DNA interaction strength via the remodeling of DNA shape.
- Photonic crystal sensors based on porous silicon. [Journal Article, Research Support, Non-U.S. Gov't]
- Sensors (Basel) 2013; 13(4):4694-713.
Porous silicon has been established as an excellent sensing platform for the optical detection of hazardous chemicals and biomolecular interactions such as DNA hybridization, antigen/antibody binding, and enzymatic reactions. Its porous nature provides a high surface area within a small volume, which can be easily controlled by changing the pore sizes. As the porosity and consequently the refractive index of an etched porous silicon layer depends on the electrochemial etching conditions photonic crystals composed of multilayered porous silicon films with well-resolved and narrow optical reflectivity features can easily be obtained. The prominent optical response of the photonic crystal decreases the detection limit and therefore increases the sensitivity of porous silicon sensors in comparison to sensors utilizing Fabry-Pérot based optical transduction. Development of porous silicon photonic crystal sensors which allow for the detection of analytes by the naked eye using a simple color change or the fabrication of stacked porous silicon photonic crystals showing two distinct optical features which can be utilized for the discrimination of analytes emphasize its high application potential.