naked DNA [keywords]
- Paper-Origami-Based Multiplexed Malaria Diagnostics from Whole Blood. [JOURNAL ARTICLE]
- Angew Chem Int Ed Engl 2016 Aug 24.
We demonstrate, for the first time, the multiplexed determination of microbial species from whole blood using the paper-folding technique of origami to enable the sequential steps of DNA extraction, loop-mediated isothermal amplification (LAMP), and array-based fluorescence detection. A low-cost handheld flashlight reveals the presence of the final DNA amplicon to the naked eye, providing a "sample-to-answer" diagnosis from a finger-prick volume of human blood, within 45 min, with minimal user intervention. To demonstrate the method, we showed the identification of three species of Plasmodium, analyzing 80 patient samples benchmarked against the gold-standard polymerase chain reaction (PCR) assay in an operator-blinded study. We also show that the test retains its diagnostic accuracy when using stored or fixed reference samples.
- PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2B(Glu2). [Journal Article]
- Nat Commun 2016.:12404.
PARP3 is a member of the ADP-ribosyl transferase superfamily that we show accelerates the repair of chromosomal DNA single-strand breaks in avian DT40 cells. Two-dimensional nuclear magnetic resonance experiments reveal that PARP3 employs a conserved DNA-binding interface to detect and stably bind DNA breaks and to accumulate at sites of chromosome damage. PARP3 preferentially binds to and is activated by mononucleosomes containing nicked DNA and which target PARP3 trans-ribosylation activity to a single-histone substrate. Although nicks in naked DNA stimulate PARP3 autoribosylation, nicks in mononucleosomes promote the trans-ribosylation of histone H2B specifically at Glu2. These data identify PARP3 as a molecular sensor of nicked nucleosomes and demonstrate, for the first time, the ribosylation of chromatin at a site-specific DNA single-strand break.
- Self-Assembled DNA Hydrogel Based on Enzymatically Polymerized DNA for Protein Encapsulation and Enzyme/DNAzyme Hybrid Cascade Reaction. [JOURNAL ARTICLE]
- ACS Appl Mater Interfaces 2016 Aug 26.
DNA hydrogel is a promising biomaterial for biological and medical applications due to its native biocompatibility and biodegradability. Herein, we provide a novel, versatile, and cost-effective approach for self-assembly of DNA hydrogel using the enzymatically polymerized DNA building blocks. The X-shaped DNA motif was elongated by terminal deoxynucleotidyl transferase (TdT) to form the building blocks, and hybridization between dual building blocks via their complementary TdT-polymerized DNA tails led to gel formation. TdT polymerization dramatically reduced the required amount of original DNA motifs, and the hybridization-mediated cross-linking of building blocks endows the gel with high mechanical strength. The DNA hydrogel can be applied for encapsulation and controllable release of protein cargos (for instance, green fluorescent protein) due to its enzymatic responsive properties. Moreover, this versatile strategy was extended to construct a functional DNAzyme hydrogel by integrating the peroxidase-mimicking DNAzyme into DNA motifs. Furthermore, a hybrid cascade enzymatic reaction system was constructed by coencapsulating glucose oxidase and β-galactosidase into DNAzyme hydrogel. This efficient cascade reaction provides not only a potential method for glucose/lactose detection by naked eye but also a promising modular platform for constructing a multiple enzyme or enzyme/DNAzyme hybrid system.
- Cascaded multiple amplification strategy for ultrasensitive detection of HIV/HCV virus DNA. [JOURNAL ARTICLE]
- Biosens Bioelectron 2016 Aug 5.:116-121.
Ultrasensitive detection of HIV and HCV virus DNA is of great importance for early accurate diagnostics and therapy of HIV virus-infected patients. Herein, to our best knowledge, it is the first to use DNA cascaded multiple amplification strategy for ultrasensitive detection of HIV virus DNA with G-quadruplex-specific fluorescent or colorimetric probes as signal carriers. The developed strategy also exhibited universal applicability for HCV virus DNA detection. After reaction for about 4h, high sensitivity and specificity can be achieved at both fluorescent and colorimetric strategies (limit of detection (LOD) of 10 fM and 0.5pM were reached for fluorescent and colorimetric detection, respectively). And the single-based mismatched DNA even can be distinguished by naked eyes. It is believed that the cascaded multiple amplification strategy presents a huge advance in sensing platform and potential application in future clinical diagnosis.
- Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing. [Journal Article]
- Sci Rep 2016.:31180.
Interleukin-8 (IL-8) gene polymorphisms have been considered as susceptibility factors in periodontal disease. However, the functional roles of IL-8 gene haplotypes have not been investigated. Here, we demonstrate for the first time the use of the CRISPR/Cas9 system to engineer the IL-8 gene, and tested the functionality of different haplotypes. Two sgRNAs vectors targeting the IL-8 gene and the naked homologous repair DNA carrying different haplotypes were used to successfully generate HEK293T cells carrying the AT genotype at the first SNP - rs4073 (alias -251), TT genotype at the second SNP - rs2227307 (alias +396), TC or CC genotypes at the third SNP - rs2227306 (alias +781) at the IL-8 locus. When stimulated with Poly I:C, ATC/TTC haplotype, cells significantly up-regulated the IL-8 at both transcriptional and translational levels. To test whether ATC/TTC haplotype is functional, we used a trans-well assay to measure the transmigration of primary neutrophils incubated with supernatants from the Poly I:C stimulation experiment. ATC/TTC haplotype cells significantly increased transmigration of neutrophils confirming the functional role for this IL-8 haplotype. Taken together, our data provides evidence that carriage of the ATC/TTC haplotype in itself may increase the influx of neutrophils in inflammatory lesions and influence disease susceptibility.
- Synthesis of a novel PEGDGA-coated hPAMAM complex as an efficient and biocompatible gene delivery vector: an in vitro and in vivo study. [JOURNAL ARTICLE]
- Drug Deliv 2016 Aug 5.:1-14.
hPAMAM/DNA polyplexes, compared to viral vectors, display unique characteristics including more safety, less immune response outcomes, a simpler synthesis and an easier process. Given the importance of these polymers, hPAMAM coated with the PEGDGA copolymer was developed as a promising non-viral gene carrier. In the present study, a new complex of hPAMAM, PEGDGA-modified hyperbranched polyamidoamine (hPAMAM), was established as a versatile non-viral gene vector. The hPAMAM polymer was synthesized by using a modified one-pot method. The resulting hPAMAM-PEGDGA polymer was able to efficiently protect encapsulated-DNA against degradation for over 2 h. In addition to low cytotoxicity, the transfection efficiency of hPAMAM-PEGDGA represented much higher (p < 0.05) than that of Lipofectamine 2000 in both MCF7 and MDA-MB231 cells (an approximately 4.5-fold increase). Cellular uptake of hPAMAM-PEGDGA in MDA-MB231 cells, 254.79 ± 2.1, was significantly higher than that in MCF7 cells, 51.61 ± 6.1 (p < 0.05). EMA-labeled DNA can be clearly observed in the tumor tissue of mice receiving hPAMAM-PEGDGA/EMA-labeled DNA. However, a significant number of fluorescent spots can be found in the tumor tissue of mice receiving hPAMAM/DNA, when compared to those treated with naked hPAMAM/DNA. It has been observed that GFP is expressed more highly in hPAMAM-PEGDGA/EMA-labeled/DNA than the one in PAMAM/DNA. The results indicated that hPAMAM-PEGDGA-mediated gene delivery to breast cancer cells is a feasible and effective strategy that may offer a new therapeutic avenue as a non-viral gene delivery carrier. Notably, According to these findings, this newly-introduced copolymer, the hPAMAM-PEGDGA complex, has proved to be a promising strategy for drug or gene delivery to tissues or cell types of interest, particularly to triple-negative breast cancer.
- Roles of long noncoding RNAs in chromosome domains. [REVIEW, JOURNAL ARTICLE]
- Wiley Interdiscip Rev RNA 2016 Aug 3.
The cell nucleus is highly organized and functionally compartmentalized. Double-stranded naked DNA is complexed with core histones and assembled into nucleosomes and chromatin, which are surrounded by nuclear domains composed of RNAs and proteins. Recently, three-dimensional views of chromosome organization beyond the level of the nucleosome have been established and are composed of several layers of chromosome domains. Only a small portion of the human genome encodes proteins; the majority is pervasively transcribed into noncoding RNAs whose functions are under intensive investigation. Importantly, the questions of how nuclear retained noncoding RNAs play roles in orchestrating the chromatin structure that have been addressed. We discuss the novel noncoding RNA clusters, Eleanors, which are derived from a large chromatin domain. They accumulate at the site of their own transcription to form RNA clouds in the nucleus, and they activate gene expression in the chromatin domain. Noncoding RNAs have emerging roles in genome regulation that are integrated into the spatial organization of chromatin and the nucleus. For further resources related to this article, please visit the WIREs website.
- Visualizing the Nonhomogeneous Structure of RAD51 Filaments Using Nanofluidic Channels. [Journal Article]
- Langmuir 2016 Aug 23; 32(33):8403-12.
RAD51 is the key component of the homologous recombination pathway in eukaryotic cells and performs its task by forming filaments on DNA. In this study we investigate the physical properties of RAD51 filaments formed on DNA using nanofluidic channels and fluorescence microscopy. Contrary to the bacterial ortholog RecA, RAD51 forms inhomogeneous filaments on long DNA in vitro, consisting of several protein patches. We demonstrate that a permanent "kink" in the filament is formed where two patches meet if the stretch of naked DNA between the patches is short. The kinks are readily seen in the present microscopy approach but would be hard to identify using conventional single DNA molecule techniques where the DNA is more stretched. We also demonstrate that protein patches separated by longer stretches of bare DNA roll up on each other and this is visualized as transiently overlapping filaments. RAD51 filaments can be formed at several different conditions, varying the cation (Mg(2+) or Ca(2+)), the DNA substrate (single-stranded or double-stranded), and the RAD51 concentration during filament nucleation, and we compare the properties of the different filaments formed. The results provide important information regarding the physical properties of RAD51 filaments but also demonstrate that nanofluidic channels are perfectly suited to study protein-DNA complexes.
- Nano-Delivery Vehicles/Adjuvants for DNA Vaccination Against HIV. [Journal Article, Research Support, Non-U.S. Gov't]
- J Nanosci Nanotechnol 2016 Mar; 16(3):2126-33.
More than 75 million people has been infected HIV and it is responsible for nearly 36 million deaths on a global scale. As one of the deadliest infectious diseases, HIV is becoming the urgent issue of the global epidemic to tackle. In order to settle this problem from the source, some effective prevention strategies should be developed to control the pandemic of HIV. Vaccines, especially DNA vaccines, could be the optimal way to control the spread of HIV due to the unparalleled superiority that DNA vaccines could generate long-term humoral and cellular immune responses which could provide protective immunity for HIV. But the naked DNA could hardly enter into cells and is easily degraded by DNases and lysosomes, so designing effective delivery system is a promising strategy. Since delivery system could be constructed to promote efficient delivery of DNA into mammalian cells, protect them from degradation, and also could be established to be a target system to arrive at certain position of expectation. The current review discusses the potential of various nano-delivery vehicles/adjuvants such as polymer, lipid, liposome, peptide and inorganic material in improving efficiency of diverse modalities available for HIV DNA vaccines.
- Age-related reduction of chromatin fractal dimension in toluidine blue - stained hepatocytes. [Journal Article]
- Mech Ageing Dev 2016 Jul.:30-4.
In this study, we proposed a hypothesis that chromatin of mouse hepatocytes exhibits age-related reduction of fractal dimension. This hypothesis was based on previously published works demonstrating that complexity of biological systems such as tissues, decreases during the process of physiological aging. Liver tissue was obtained from 24 male mice divided into 3 age groups: 10-days-old (young, juvenile), 210-days-old (adult) and 390-days-old. The tissue was stained using a modification of toluidine blue (nucleic acid - specific) staining method. A total of 480 chromatin structures (20 for each animal) were analyzed. For each structure, the values of fractal dimension, lacunarity, textural angular second moment and inverse difference moment were calculated using ImageJ software and its plugins. The results indicated the age-related reduction in fractal dimension and increase in lacunarity (p<0.01). Fractal dimension is a potentially good indicator of age associated changes in chromatin structure. To our knowledge, this is the first study to show that fractal complexity of hepatocyte chromatin decreases during the process of physiological aging. Fractal analysis as a method could be useful in detection of small age-related changes in chromatin distribution not otherwise visible with naked eye on conventional tissue micrographs.