Intelligent Reconfiguration-Promoted Cellular Internalization of Core-Shell DNA Nanoprobe Equipped with Successive Dual Stimuli-Responsive Protective Satellites for Amplification Fluorescence Imaging of Tumor Cells.

Although DNA probes have attracted increasing interest for precise tumor cell identification by imaging intracellular biomarkers, the requirement of commercial transfection reagents, limited targeting ligands, and/or non-biocompatible inorganic nanostructures has hampered the clinic translation. To circumvent these shortcomings, a reconfigurable ES-NC (Na+-dependent DNAzyme (E)-based substrate (S) cleavage core/shell DNA nanocluster (NC)) entirely from DNA strands is assembled for precise imaging of cancerous cells in a successive dual-stimuli-responsive manner. This nanoprobe is composed of a strung DNA tetrahedral satellites-based protective (DTP) shell, parallelly aligned target-responsive sensing (PTS) interlayer, and hydrophobic cholesterol-packed innermost layer (HCI core). Tetrahedral axial rotation-activated reconfiguration of DTP shell promotes the exposure of interior hydrophobic moieties, enabling cholesterol-mediated cellular internalization without auxiliary elements. Within cells, over-expressed glutathione triggers the disassembly of the DTP protective shell (first stimulus), facilitating target-stimulated signal transduction/amplification process (second stimuli). Target miRNA-21 is detected down to 10.6 fM without interference from coexisting miRNAs. Compared with transfection reagent-mediated counterpart, ES-NC displays a higher imaging ability, resists nuclease degradation, and has no detectable damage to healthy cells. The blind test demonstrates that the ES-NC is suitable for the identification of cancerous cells from healthy cells, indicating a promising tool for early diagnosis and prediction of cancer.

Accurate SNV detection in single cells by transposon-based whole-genome amplification of complementary strands.

Single-nucleotide variants (SNVs), pertinent to aging and disease, occur sporadically in the human genome, hence necessitating single-cell measurements. However, detection of single-cell SNVs suffers from false positives (FPs) due to intracellular single-stranded DNA damage and the process of whole-genome amplification (WGA). Here, we report a single-cell WGA method termed multiplexed end-tagging amplification of complementary strands (META-CS), which eliminates nearly all FPs by virtue of DNA complementarity, and achieved the highest accuracy thus far. We validated META-CS by sequencing kindred cells and human sperm, and applied it to other human tissues. Investigation of mature single human neurons revealed increasing SNVs with age and potentially unrepaired strand-specific oxidative guanine damage. We determined SNV frequencies along the genome in differentiated single human blood cells, and identified cell type-dependent mutational patterns for major types of lymphocytes.

DNA-Based Gold Nanoparticle Assemblies: From Structure Constructions to Sensing Applications.

Gold nanoparticles (Au NPs) have become one of the building blocks for superior assembly and device fabrication due to the intrinsic, tunable physical properties of nanoparticles. With the development of DNA nanotechnology, gold nanoparticles are organized in a highly precise and controllable way under the mediation of DNA, achieving programmability and specificity unmatched by other ligands. The successful construction of abundant gold nanoparticle assembly structures has also given rise to the fabrication of a wide range of sensors, which has greatly contributed to the development of the sensing field. In this review, we focus on the progress in the DNA-mediated assembly of Au NPs and their application in sensing in the past five years. Firstly, we highlight the strategies used for the orderly organization of Au NPs with DNA. Then, we describe the DNA-based assembly of Au NPs for sensing applications and representative research therein. Finally, we summarize the advantages of DNA nanotechnology in assembling complex Au NPs and outline the challenges and limitations in constructing complex gold nanoparticle assembly structures with tailored functionalities.

Direct visualization of local activities of long DNA strands via image-time correlation.

Bacteriophages with long DNA genomes are of interest due to their diverse mutations dependent on environmental factors. By lowering the ionic strength of a hydrophobic (PPh4Cl) antagonistic salt (at 1 mM), single long T4 DNA strand fluctuations were clearly observed, while condensed states of T4 DNA globules were formed above 5-10 mM salt. These long DNA strands were treated with fluorescently labeled probes, for which photo bleaching is often unavoidable over a short time of measurement. In addition, long (few tens of [Formula: see text]) length scales are required to have larger fields of view for better sampling, with shorter temporal resolutions. Thus, an optimization between length and time is crucial to obtain useful information. To facilitate the challenge of detecting large biomacromolecules, we here introduce an effective method of live image data analysis for direct visualization and quantification of local thermal fluctuations. The motions of various conformations for the motile long DNA strands were examined for the single- and multi-T4 DNA strands. We find that the unique correlation functions exhibit a relatively high-frequency oscillatory behavior superimposed on the overall slower decay of the correlation function with a splitting of amplitudes deriving from local activities of the long DNA strands. This work shows not only the usefulness of an image-time correlation for analyzing large biomacromolecules, but also provides insight into the effects of a hydrophobic antagonistic salt on active T4 bacteriophage long DNA strands, including thermal translocations in their electrostatic interactions.

Melatonin attenuates 60 Co γ-ray-induced hematopoietic, immunological and gastrointestinal injuries in C57BL/6 male mice.

Protection of hematopoietic, immunological, and gastrointestinal injuries from deleterious effects of ionizing radiation is prime rational for developing radioprotector. The objective of this study, therefore, was to evaluate the radioprotective potential of melatonin against damaging effects of radiation-induced hematopoietic, immunological, and gastrointestinal injuries in mice. C57BL/6 male mice were intraperitoneally administered with melatonin (50-150 mg/kg) 30 min prior to whole-body radiation exposure of 5 and 7.5 Gy using 60 Co-teletherapy unit. Thirty-day survival against 7.5 Gy was monitored. Melatonin (100 mg/kg) pretreatment showed 100% survival against 7.5 Gy radiation dose. Melatonin pretreatment expanded femoral HPSCs, and inhibited spleenocyte DNA strands breaks and apoptosis in irradiated mice. At this time, it also protected radiation-induced loss of T cell sub-populations in spleen. In addition, melatonin pretreatment enhanced crypts regeneration and increased villi number and length in irradiated mice. Translocation of gut bacteria to spleen, liver and kidney were controlled in irradiated mice pretreated with melatonin. Radiation-induced gastrointestinal DNA strand breaks, lipid peroxidation, and expression of proapoptotic-p53, Bax, and antiapoptotic-Bcl-xL proteins were reversed in melatonin pretreated mice. This increase of Bcl-xL was associated with the decrease of Bax/Bcl-xL ratio. ABTS and DPPH radical assays revealed that melatonin treatment alleviated total antioxidant capacity in hematopoietic and gastrointestinal tissues. Present study demonstrated that melatonin pretreatment was able to prevent hematopoietic, immunological, and gastrointestinal radiation-induced injury, therefore, overcoming lethality in mice. These results suggest potential of melatonin in developing radioprotector for protection of bone marrow, spleen, and gastrointestine in planned radiation exposure scenarios including radiotherapy. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 501-518, 2017.

Twist versus nonlinear stacking in short DNA molecules.

The denaturation of the double helix is a template for fundamental biological functions such as replication and transcription involving the formation of local fluctuational openings. The denaturation transition is studied for heterogeneous short sequences of DNA, i.e. ~100 base pairs, in the framework of a mesoscopic Hamiltonian model which accounts for the helicoidal geometry of the molecule. The theoretical background for the application of the path integral formalism to predictive analysis of the molecule thermodynamical properties is discussed. The base pair displacements with respect to the ground state are treated as paths whose temperature dependent amplitudes are governed by the thermal wavelength. The ensemble of base pairs paths is selected, at any temperature, consistently with both the model potential and the second law of thermodynamics. The partition function incorporates the effects of the base pair thermal fluctuations which become stronger close to the denaturation. The transition appears as a gradual phenomenon starting from the molecule segments rich in adenine-thymine base pairs. Computing the equilibrium thermodynamics, we focus on the interplay between twisting of the complementary strands around the molecule axis and nonlinear stacking potential: it is shown that the latter affects the melting profiles only if the rotational degrees of freedom are included in the Hamiltonian. The use of ladder Hamiltonian models for the DNA complementary strands in the pre-melting regime is questioned.

Inhibition of kinetic random-distribution in DNA Seesaw gates and biosensors for complete leakage prevention.

DNA nanomaterials have a wide application prospect in biomedical field, among which DNA computers and biosensors based on Seesaw-based DNA circuit is considered to have the most development potential. However, the serious leakage of Seesaw-based DNA circuit prevented its further development and application. Moreover, the existing methods to suppress leakage can't achieve the ideal effect. Interestingly, we found a new source of leakage in Seesaw-based DNA circuit, which we think is the main reason why the previous methods to suppress leakage are not satisfactory. Therefore, based on this discovery, we use DNA triplex to design a new method to suppress the leakage of Seesaw-based DNA circuit. Its ingenious design makes it possible to perfectly suppress the leakage of all sources in Seesaw-based DNA circuit and ensure the normal output of the circuit. Based on this technology, we have constructed basic Seesaw module, AND gate, OR gate, secondary complex circuits and DNA detector. Experimental results show that we can increase the working range of the secondary Seesaw-based DNA circuit by five folds and keep its normal output signal above 90%, and we can improve the LOD of the Seesaw-based DNA detector to 1/11 of the traditional one(1.8pM). More importantly, we successfully developed a detector with adjustable detection range, which can theoretically achieve accurate detection in any concentration range. We believe the established triplex blocking strategy will greatly facilitate the most powerful Seesaw based DNA computers and biosensors, and further promote its application in biological systems.

Mathematical formalism of femtosecond laser-deoxyribonucleic acid interaction: thermal evolution.

A novel analytical formalism is proposed based upon Quantum heat transport equation in order to describe the femtoseconds/picoseconds laser pulses interaction with the Deoxyribonucleic acid (DNA). The formalism generates solutions based upon inputs as: voltage, laser beam intensity and laser - DNA interaction time. Thermal waves induced inside irradiated DNA are defined and accounted for. Analytical simulations show that the optimum regime of laser - DNA interaction was reached for a potential carrier generated at the interface equal to 3.5 × 10-3 eV. It has to be mentioned that the formalism breaks down if the potential carrier generated at the interface is inferior to 10-2 eV. Accordingly, for pulse duration inferior to 1 ps, the laser beam spatial-temporal distribution has an essential role in defining the shape and magnitude of the thermal distribution within the irradiated DNA strands.

Identification of genetic instability in peripheral blood lymphocyte of oral squamous cell carcinoma patients assess by comet assay.

Context: Studies established that human cancer is principally a genetic disease; it arises as accumulation of a set of genetic changes. In the pathogenesis of cancer, genetic instability is the sequential event to a carcinogenic stimulus resulting in various genomic changes including DNA damage. Aims: To assess genetic instability, as susceptibility to DNA damage, we used single-cell gel electrophoresis (comet assay) to study double strand breaks in associated with the risk of oral squamous cell carcinoma (OSCC). Materials and Methods: We used comet assay to measure double strand break in individual peripheral blood lymphocytes from 50 individuals with OSCC and 30 healthy control subjects. All personal information was gathered from subjects including tobacco history. DNA damage was visualized as comet assay and quantified by movement of damaged strands as length of tail. Results: Study results of OSCC patients were observed in relation to clinical staging and histological grading of carcinoma. On the basis of clinical observation, cases were grouped in to Stage I, Stage II, Stage III and Stage IV. No stage I cases were in study sample. The mean DNA damage migration length was observed 4.600 ± 0.4613 µm in stage II, whereas in Stage III and Stage IV, it was observed to be 4.961 ± 0.5620 µm and 4.883 ± 0.410 µm, respectively. The DNA damage length in histological grades of squamous cell carcinoma patients in Grade I was 4.6437 ± 0.3061 µm and Grade II was 5.3533 ± 0.3831 µm. In comparison with control group and squamous cell carcinoma group, it was observed in the range of 0.02-0.36 µm and varied from 4.04 to 5.84 µm range, respectively. Thus, the results were statistically significant with the histological grading of OSCC. Statistical Analysis: Unpaired' test and "ANOVA" test are used for statistics. Statistical Analysis: Unpaired' test and "ANOVA" test are used for statistics. Conclusion: The amount of DNA strand breaks in peripheral lymphocytes are measured by comet assay which is associated with relative risk of OSCC.

DNA strand patterns on aluminium thin films.

A new patterning method using Deoxyribose Nucleic Acid (DNA) strands capable of producing nanogaps of less than 100 nm is proposed and investigated in this work. DNA strands from Bosenbergia rotunda were used as the fundamental element in patterning DNA on thin films of aluminium (Al) metal without the need for any lithographic techniques. The DNA strands were applied in buffer solutions onto thin films of Al on silicon (Si) and the chemical interactions between the DNA strands and Al creates nanometer scale arbitrary patterning by direct transfer of the DNA strands onto the substrate. This simple and cost-effective method can be utilized in the fabrication of various components in electronic chips for microelectronics and Nano Electronic Mechanical System (NEMS) applications in general.

Feasibility studies on si-based biosensors.

The aim of this paper is to summarize the efforts carried out so far in the fabrication of Si-based biosensors by a team of researchers in Catania, Italy. This work was born as a collaboration between the Catania section of the Microelectronic and Microsystem Institute (IMM) of the CNR, the Surfaces and Interfaces laboratory (SUPERLAB) of the Consorzio Catania Ricerche and two departments at the University of Catania: the Biomedical Science and the Biological Chemistry and Molecular Biology Departments. The first goal of our study was the definition and optimization of an immobilization protocol capable of bonding the biological sensing element on a Si-based surface via covalent chemical bonds. We chose SiO(2) as the anchoring surface due to its biocompatibility and extensive presence in microelectronic devices. The immobilization protocol was tested and optimized, introducing a new step, oxide activation, using techniques compatible with microelectronic processing. The importance of the added step is described by the experimental results. We also tested different biological molecule concentrations in the immobilization solutions and the effects on the immobilized layer. Finally a MOS-like structure was designed and fabricated to test an electrical transduction mechanism. The results obtained so far and the possible evolution of the research field are described in this review paper.

Supercoil Levels in E. coli and Salmonella Chromosomes Are Regulated by the C-Terminal 35⁻38 Amino Acids of GyrA.

Prokaryotes have an essential gene-gyrase-that catalyzes negative supercoiling of plasmid and chromosomal DNA. Negative supercoils influence DNA replication, transcription, homologous recombination, site-specific recombination, genetic transposition and sister chromosome segregation. Although E. coli and Salmonella Typhimurium are close relatives with a conserved set of essential genes, E. coli DNA has a supercoil density 15% higher than Salmonella, and E. coli cannot grow at the supercoil density maintained by wild type (WT) Salmonella. E. coli is addicted to high supercoiling levels for efficient chromosomal folding. In vitro experiments were performed with four gyrase isoforms of the tetrameric enzyme (GyrA2:GyrB2). E. coli gyrase was more processive and faster than the Salmonella enzyme, but Salmonella strains with chromosomal swaps of E. coli GyrA lost 40% of the chromosomal supercoil density. Reciprocal experiments in E. coli showed chromosomal dysfunction for strains harboring Salmonella GyrA. One GyrA segment responsible for dis-regulation was uncovered by constructing and testing GyrA chimeras in vivo. The six pinwheel elements and the C-terminal 35⁻38 acidic residues of GyrA controlled WT chromosome-wide supercoiling density in both species. A model of enzyme processivity modulated by competition between DNA and the GyrA acidic tail for access to ß-pinwheel elements is presented.

Hairpin Bisulfite Sequencing: Synchronous Methylation Analysis on Complementary DNA Strands of Individual Chromosomes.

The accurate and quantitative detection of 5-methylcytosine is of great importance in the field of epigenetics. The method of choice is usually bisulfite sequencing because of the high resolution and the possibility to combine it with next generation sequencing. Nevertheless, also this method has its limitations. Following the bisulfite treatment DNA strands are no longer complementary such that in a subsequent PCR amplification the DNA methylation patterns information of only one of the two DNA strand is preserved. Several years ago Hairpin Bisulfite sequencing was developed as a method to obtain the pattern information on complementary DNA strands. The method requires fragmentation (usually by enzymatic cleavage) of genomic DNA followed by a covalent linking of both DNA strands through ligation of a short DNA hairpin oligonucleotide to both strands. The ligated covalently linked dsDNA products are then subjected to a conventional bisulfite treatment during which all unmodified cytosines are converted to uracils. During the treatment the DNA is denatured forming noncomplementary ssDNA circles. These circles serve as a template for a locus specific PCR to amplify chromosomal patterns of the region of interest. As a result one ends up with a linearized product, which contains the methylation information of both complementary DNA strands.

Detection of DNA damage based on metal-mediated molecular beacon and DNA strands displacement reaction.

DNA hairpin structure probes are usually designed by forming intra-molecular duplex based on Watson-Crick hydrogen bonds. In this paper, a molecular beacon based on silver ions-mediated cytosine-Ag(+)-cytosine base pairs was used to detect DNA. The inherent characteristic of the metal ligation facilitated the design of functional probe and the adjustment of its binding strength compared to traditional DNA hairpin structure probes, which make it be used to detect DNA in a simple, rapid and easy way with the help of DNA strands displacement reaction. The method was sensitive and also possesses the good specificity to differentiate the single base mismatched DNA from the complementary DNA. It was also successfully applied to study the damage effect of classic genotoxicity chemicals such as styrene oxide and sodium arsenite on DNA, which was significant in food science, environmental science and pharmaceutical science.

The use of complimentary assays to evaluate the enrichment of human sperm quality in asthenoteratozoospermic and teratozoospermic samples processed with Annexin-V magnetic activated cell sorting.

Sperm chromatin integrity may affect the outcomes of assisted reproductive technology (ART). Developing a clinically reliable strategy to enrich sperm samples with high chromatin quality spermatozoa prior to sperm banking or use in ART would thus be advantageous. The objectives of this study were to: (i) assess the sperm chromatin quality in men with different categories of semen parameters; and (ii) evaluate the extents of Annexin-V magnetic-activated cell sorting (MACS) technology coupled with differential density gradient centrifugation (DGC) in improving sperm chromatin quality. Three categories of men from couples attending a university-based fertility clinic were recruited based on their semen parameters: normozoospermic (n = 13), asthenoteratozoospermic (n = 17) and teratozoospermic (n = 12). For each patient, spermatozoa in semen samples were processed first by DGC to enrich the motility and further by MACS to remove spermatozoa showing apoptotic features. The yield and enrichment of sperm quality was evaluated at each step with conventional semen parameters in conjunction with a combination of five complementary assays, to assess sperm maturity, chromatin structure, compaction and DNA integrity (Hyaluronic Binding Assay, SCSA, chromomycine A3 staining and TUNEL and COMET assays). Our results demonstrated that, compared with normozoospermic samples, raw asthenoteratozoospermic and teratozoospermic samples had a higher proportion of spermatozoa containing DNA breaks, but only asthenoteratozoospermic exhibited altered chromatin structure and decreased binding to hyaluronic acid. Interestingly, the DGC appeared to select for more mature spermatozoa with high DNA compaction. More importantly, in all categories of semen samples, Annexin-V MACS allows enrichment of spermatozoa with good chromatin quality as measured by the TUNEL and SCSA. Because effective treatment modalities to improve sperm DNA damage are limited, our results suggest a potential clinical value of MACS as a mean to enhance sperm quality that may improve assisted reproductive outcomes.

Left-right symmetry breaking in mice by left-right dynein may occur via a biased chromatid segregation mechanism, without directly involving the Nodal gene.

Ever since cloning the classic iv (inversedviscerum) mutation identified the "left-right dynein" (lrd) gene in mice, most research on body laterality determination has focused on its function in motile cilia at the node embryonic organizer. This model is attractive, as it links chirality of cilia architecture to asymmetry development. However, lrd is also expressed in blastocysts and embryonic stem cells, where it was shown to bias the segregation of recombined sister chromatids away from each other in mitosis. These data suggested that lrd is part of a cellular mechanism that recognizes and selectively segregates sister chromatids based on their replication history: old "Watson" versus old "Crick" strands. We previously proposed that the mouse left-right axis is established via an asymmetric cell division prior to/or during gastrulation. In this model, left-right dynein selectively segregates epigenetically differentiated sister chromatids harboring a hypothetical "left-right axis development 1" ("lra1") gene during the left-right axis establishing cell division. Here, asymmetry development would be ultimately governed by the chirality of the cytoskeleton and the DNA molecule. Our model predicts that randomization of chromatid segregation in lrd mutants should produce embryos with 25% situs solitus, 25% situs inversus, and 50% embryonic death due to heterotaxia and isomerism. Here we confirmed this prediction by using two distinct lrd mutant alleles. Other than lrd, thus far Nodal gene is the most upstream function implicated in visceral organs laterality determination. We next tested whether the Nodal gene constitutes the lra1 gene hypothesized in the model by testing mutant's effect on 50% embryonic lethality observed in lrd mutants. Since Nodal mutation did not suppress lethality, we conclude that Nodal is not equivalent to the lra1 gene. In summary, we describe the origin of 50% lethality in lrd mutant mice not yet explained by any other laterality-generating hypothesis.