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Ineke Brouwer, Hongshan Zhang, Andrea Candelli, Davide Normanno, Erwin J G Peterman, Gijs J L Wuite, Mauro Modesti
Human RAD52 promotes annealing of complementary single-stranded DNA (ssDNA). In-depth knowledge of RAD52-DNA interaction is required to understand how its activity is integrated in DNA repair processes. Here, we visualize individual fluorescent RAD52 complexes interacting with single DNA molecules. The interaction with ssDNA is rapid, static, and tight, where ssDNA appears to wrap around RAD52 complexes that promote intra-molecular bridging. With double-stranded DNA (dsDNA), interaction is slower, weaker, and often diffusive...
March 21, 2017: Cell Reports
Dongshan Xiang, Kun Zhai, Qiuzhang Sang, Boan Shi, Xiaohong Yang
A highly sensitive and selective fluorescence method of quantitative detection for mercury in soil was developed using non-labeled molecular beacon (MB), single-stranded nucleic acid (ssDNA) and fluorescent dye Hoechst 33258. In this analytical method, the loop of MB was designed to be a sequence that was complementary to the ssDNA with multiple T-T mismatches, the stem of MB was completely designed as C-G base pairs, and both ends of the MB are not modified by any fluorophore and quencher. In the absence of Hg(2+), the interaction between Hoechst 33258 and the MB was very weak, and the fluorescence signal of Hoechst 33258 was very low...
2017: Analytical Sciences: the International Journal of the Japan Society for Analytical Chemistry
Hadar Ben-Yoav, Peter H Dykstra, William E Bentley, Reza Ghodssi
A microfluidic electrochemical lab-on-a-chip (LOC) device for DNA hybridization detection has been developed. The device comprises a 3 × 3 array of microelectrodes integrated with a dual layer microfluidic valved manipulation system that provides controlled and automated capabilities for high throughput analysis of microliter volume samples. The surface of the microelectrodes is functionalized with single-stranded DNA (ssDNA) probes which enable specific detection of complementary ssDNA targets. These targets are detected by a capacitive technique which measures dielectric variation at the microelectrode-electrolyte interface due to DNA hybridization events...
2017: Methods in Molecular Biology
Weixin Qian, Kentaro Doi, Satoyuki Kawano
Electrokinetic phenomena in micro/nanofluidic channels have attracted considerable attention because precise control of molecular transport in liquids is required to optically and electrically capture the behavior of single molecules. However, the detailed mechanisms of polymer transport influenced by electroosmotic flows and electric fields in micro/nanofluidic channels have not yet been elucidated. In this study, a Langevin dynamics simulation was used to investigate the electrokinetic transport of single-stranded DNA (ssDNA) in a cylindrical nanochannel, employing a coarse-grained bead-spring model that quantitatively reproduced the radius of gyration, diffusion coefficient, and electrophoretic mobility of the polymer...
March 14, 2017: Biophysical Journal
E Pastuszek, J Kiewisz, P Skowronska, J Liss, M Lukaszuk, A Bruszczynska, G Jakiel, K Lukaszuk
Presence of vacuoles and degree of sperm DNA damage are considered to be the basic factors used for the assessment of sperm fertilization capacity. We aimed to investigate the link between these two parameters. According to our knowledge, this is the first study where the Comet assay was used to assess the degree of DNA fragmentation of sperm categorized by Motile Sperm Organelle Morphology Examination (MSOME) Grades. Semen samples from 10 patients were assessed. Spermatozoa were graded into four MSOME groups according to the Vanderzwalmen's criteria...
March 2017: Andrology
Wouter Engelen, Maarten Merkx
Synthetic protein switches that sequence-specifically respond to oligonucleotide-based input triggers provide valuable tools for the readout of oligonucleotide-based biomolecular systems and networks. Here, we discuss a highly modular approach to reversibly control the DNA-directed assembly and disassembly of a complex between TEM1-β-lactamase and its inhibitor protein BLIP. By conjugating each protein to a unique handle oligonucleotide, the enzyme-inhibitor pair is noncovalently assembled upon the addition of a complementary ssDNA template strand, resulting in inhibition of enzyme activity...
2017: Methods in Molecular Biology
Do-Kyun Kim, Soo-Ji Kim, Dong-Hyun Kang
In order to assure the microbial safety of drinking water, UVC-LED treatment has emerged as a possible technology to replace the use of conventional low pressure (LP) mercury vapor UV lamps. In this investigation, inactivation of Human Enteric Virus (HuEV) surrogates with UVC-LEDs was investigated in a water disinfection system, and kinetic model equations were applied to depict the surviving infectivities of the viruses. MS2, Qβ, and ΦX 174 bacteriophages were inoculated into sterile distilled water (DW) and irradiated with UVC-LED printed circuit boards (PCBs) (266nm and 279nm) or conventional LP lamps...
January 2017: Food Research International
Casey Tyler Andrews, Brady A Campbell, Adrian Hamilton Elcock
Given the ubiquitous nature of protein-DNA interactions, it is important to understand the interaction thermodynamics of individual amino acid sidechains for DNA. One way to assess these preferences is to perform molecular dynamics (MD) simulations. Here we report MD simulations of twenty amino acid sidechain analogs interacting simultaneously with both a 70-base pair double-stranded DNA and with a 70-nucleotide single-stranded DNA. The relative preferences of the amino acid sidechains for dsDNA and ssDNA match well with values deduced from crystallographic analyses of protein-DNA complexes...
March 13, 2017: Journal of Chemical Theory and Computation
Joaquim S L Vong, Jason C H Tsang, Peiyong Jiang, Wing-Shan Lee, Tak Yeung Leung, K C Allen Chan, Rossa W K Chiu, Y M Dennis Lo
BACKGROUND: Recent studies have suggested that single-stranded DNA (ssDNA) library preparation can enrich short DNA species from the plasma of healthy individuals, cancer patients, and transplant recipients. Based on previous observations that fetal DNA molecules in the maternal plasma are shorter than maternal DNA molecules, ssDNA library preparation may potentially enrich fetal DNA and provide substantial improvement in noninvasive prenatal testing. METHODS: We tested this hypothesis by comparing the maternal plasma DNA sequencing results using 2 types of ssDNA library preparation methods and a standard double-stranded DNA (dsDNA) library method using samples from first- and third-trimester pregnancies...
March 9, 2017: Clinical Chemistry
Erkin Erdal, Syed Haider, Jan Rehwinkel, Adrian L Harris, Peter J McHugh
Radiotherapy and chemotherapy are effective treatment methods for many types of cancer, but resistance is common. Recent findings indicate that antiviral type I interferon (IFN) signaling is induced by these treatments. However, the underlying mechanisms still need to be elucidated. Expression of a set of IFN-stimulated genes comprises an IFN-related DNA damage resistance signature (IRDS), which correlates strongly with resistance to radiotherapy and chemotherapy across different tumors. Classically, during viral infection, the presence of foreign DNA in the cytoplasm of host cells can initiate type I IFN signaling...
March 9, 2017: Genes & Development
Wei Shi, Therese Vu, Didier Boucher, Anna Biernacka, Jules Nde, Raj K Pandita, Jasmin Straube, Glen M Boyle, Fares Al-Ejeh, Purba Nag, Jessie Jeffery, Janelle L Harris, Amanda L Bain, Marta Grzelak, Magdalena Skrzypczak, Abhishek Mitra, Norbert Dojer, Nicola Crosetto, Nicole Cloonan, Olivier J Becherel, John Finnie, Jeffrey R Skaar, Carl R Walkley, Tej K Pandita, Maga Rowicka, Krzysztof Ginalski, Steven W Lane, Kum Kum Khanna
Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The two single-stranded DNA binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however their overlapping roles during normal physiology are incompletely understood. We generated mice where both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, while conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double strand breaks (DSBs) enriched at Ssb binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA...
March 7, 2017: Blood
Wen-Qiang Wu, Xi-Miao Hou, Bo Zhang, Philippe Fossé, Brigitte René, Olivier Mauffret, Ming Li, Shuo-Xing Dou, Xu-Guang Xi
Werner syndrome is caused by mutations in the WRN gene encoding WRN helicase. A knowledge of WRN helicase's DNA unwinding mechanism in vitro is helpful for predicting its behaviors in vivo, and then understanding their biological functions. In the present study, for deeply understanding the DNA unwinding mechanism of WRN, we comprehensively characterized the DNA unwinding properties of chicken WRN helicase core in details, by taking advantages of single-molecule fluorescence resonance energy transfer (smFRET) method...
March 7, 2017: Scientific Reports
Bin Zhu, Longfei Wang, Hitoshi Mitsunobu, Xueling Lu, Alfredo J Hernandez, Yukari Yoshida-Takashima, Takuro Nunoura, Stanley Tabor, Charles C Richardson
A DNA polymerase is encoded by the deep-sea vent phage NrS-1. NrS-1 has a unique genome organization containing genes that are predicted to encode a helicase and a single-stranded DNA (ssDNA)-binding protein. The gene for an unknown protein shares weak homology with the bifunctional primase-polymerases (prim-pols) from archaeal plasmids but is missing the zinc-binding domain typically found in primases. We show that this gene product has efficient DNA polymerase activity and is processive in DNA synthesis in the presence of the NrS-1 helicase and ssDNA-binding protein...
March 6, 2017: Proceedings of the National Academy of Sciences of the United States of America
Youshen Wu, Jiajun Liu, Ya Wang, Ke Li, Lei Li, Jianhua Xu, Daocheng Wu
Novel ratiometric fluorescent short single-stranded DNA (ssDNA) nanothermometers (ssDNA FT) were developed using the fluorescence resonance energy transfer (FRET) effect of the ssDNA's end labeled fluorophores. An optimal ssDNA sequence and associated ssDNA FT were determined through combined MD simulation and temperature-related FRET analysis. Their fluorescence properties and thermo-responsivities were analyzed using fluorescence spectra. The influences of ssDNA' sequence length, sequence composition and fluorescent labels for temperature sensing were investigated...
March 14, 2017: ACS Applied Materials & Interfaces
Hai Dang Nguyen, Tribhuwan Yadav, Sumanprava Giri, Borja Saez, Timothy A Graubert, Lee Zou
R loop, a transcription intermediate containing RNA:DNA hybrids and displaced single-stranded DNA (ssDNA), has emerged as a major source of genomic instability. RNaseH1, which cleaves the RNA in RNA:DNA hybrids, plays an important role in R loop suppression. Here we show that replication protein A (RPA), an ssDNA-binding protein, interacts with RNaseH1 and colocalizes with both RNaseH1 and R loops in cells. In vitro, purified RPA directly enhances the association of RNaseH1 with RNA:DNA hybrids and stimulates the activity of RNaseH1 on R loops...
March 2, 2017: Molecular Cell
Matthew D Moore, Blanca I Escudero-Abarca, Lee-Ann Jaykus
Nucleic acid aptamers are a class of alternative ligands increasingly growing in importance in the face of contemporary detection challenges. Aptamers offer multiple advantages over traditional ligands like antibodies; however, their ability to specifically bind target molecules must first be confirmed after their generation. Use of a plate-based enzyme-linked aptamer sorbent assay (ELASA) is a generally rapid way to screen and characterize aptamer binding to protein targets. ELASA involves directly plating a protein target onto a nonspecific (polystyrene) surface and assessing binding of functionalized (biotinylated) aptamers to those plated proteins using an enzyme conjugate that recognizes the aptamers...
2017: Methods in Molecular Biology
Audrey Ric, Varravaddheay Ong-Meang, Véréna Poinsot, Nathalie Martins-Froment, Fabien Chauvet, Audrey Boutonnet, Frédéric Ginot, Vincent Ecochard, Laurent Paquereau, François Couderc
Tris-Acetate buffer is currently used in the selection and the characterization of ssDNA by capillary electrophoresis (CE). By applying high voltage, the migration of ionic species into the capillary generates a current that induces water electrolysis. This phenomenon is followed by the modification of the pH and the production of Tris derivatives. By injecting 10 times by capillary electrophoresis ssDNA (50nM), the whole oligonucleotide was degraded. In this paper, we will show that the Tris buffer in the running vials is modified along the electrophoretic process by electrochemical reactions...
March 2, 2017: Electrophoresis
Xiangjun Liu, Tao Bing, Dihua Shangguan
We present a novel microbead-based detection platform as a simple and universal strategy for simultaneous determination of multiple biomolecules. This platform is composed of streptavidin coated uniform-sized polystyrene microbeads, dye and biotin-labeled ssDNA or aptamer probes, and quencher-labeled complementary sequences. By this method, upon target binding to the probes, quencher strand dissociation is triggered, which results in fluorescence reactivation of the microbead linked probes. The fluorescence variation is readily monitored by flow cytometry and with a high sensitivity...
March 22, 2017: ACS Applied Materials & Interfaces
Erika Hasuike, Aya Mizutani Akimoto, Reiko Kuroda, Ko Matsukawa, Yuki Hiruta, Hideko Kanazawa, Ryo Yoshida
The novel property of reversible regulation of parallel type G-quadruplexes with moderate temperature changes was discovered in thermoresponsive hydrogels using a universal chiroptical spectrophotometer 1 (UCS-1). This study is the first to report the regulation of the ssDNA steric structure by using a poly(N-isopropylacrylamide) (PNIPAAm) gel.
March 1, 2017: Chemical Communications: Chem Comm
Xi-Yao Huang, Abdur Rahman Ansari, Hai-Bo Huang, Xing Zhao, Ning-Ya Li, Zhi-Jian Sun, Ke-Mei Peng, Juming Zhong, Hua-Zhen Liu
BACKGROUND: Lipopolysaccharide (LPS) induces acute liver injury and the complex mechanisms include the activation of toll like receptor 4 (TLR4) signaling pathway in many species. However, immuno-pathological changes during TLR4 signaling under LPS stress in acute liver injury is poorly understood in avian species. The present investigation was therefore carried out to evaluate these alterations in TLR4 signaling pathway during acute liver injury in young chickens. RESULTS: After intraperitoneal injection of LPS or saline, liver samples were harvested at 0, 2, 6, 12, 24, 36, 72 and 120 h (n = 6 at each time point) and the microstructures were analyzed by hematoxylin and eosin (H&E) staining...
February 27, 2017: BMC Immunology
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