Enzymatic Degradation of DNA Probed by In Situ X-ray Scattering
Abstract not provided
Krishnamoorthy, Kurinji↗
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Direct fluorescence in situ hybridization on human metaphase chromosomes using quantum dot-platinum labeled DNA probes
The telomere shortening in chromosomes implies the senescence, apoptosis, or oncogenic transformation of cells. Since detecting telomeres in aging and diseases like cancer, is important, the direct detection of telomeres has been a very useful biomarker. We propose a telomere detection method using a newly synthesized quantum dot (QD) based probe with oligonucleotide conjugation and direct fluorescence in situ hybridization (FISH). QD-oligonucleotides were prepared with metal coordination bonding based on platinum-guanine binding reported in our previous work. The QD-oligonucleotide conjugation method has an advantage where any sequence containing guanine at the end can be easily bound to the starting QD-Pt conjugate. A synthesized telomeric oligonucleotide was bound to the QD-Pt conjugate successfully and this probe hybridized specifically on the telomere of fabricated MV-4-11 and MOLT-4 chromosomes. Additionally, the QD-telomeric oligonucleotide probe successfully detected the telomeres on the CGH metaphase slide. Due to the excellent photostability and high quantum yield of QDs, the QD-oligonucleotide probe has high fluorescence intensity when compared to the organic dye-oligonucleotide probe. Our QD-oligonucleotide probe, conjugation method of this QD probe, and hybridization protocol with the chromosomes can be a useful tool for chromosome painting and FISH. - Highlights: • We prepared a probe linked between QD and telomeric oligonucleotide with platinum-guanine bonding. • Telomeres were detected by our new telomere probes successfully in three different human metaphase chromosomes. • QDPt-DNA probe has high fluorescence intensity in comparison with organic dye-DNA probe.
60 APPLIED LIFE SCIENCES↗
High Performance DNA Probes for Perinatal Detection of Numerical Chromosome Aberrations
Human reproduction is a tightly controlled process of stepwise evolution with multiple, mostly yet unknown milestones and checkpoints. Healthy halpoid gametes have to be produced by the parents, which will fuse to form the diploid zygote that implants in the female uterus and grows to become first an embryo, then a fetus and finally matures into a newborn. There are several known risk factors that interfere with normal production of gametes, spermatocytes or oocytes, and often cause embryonic mortality and fetal demise at an early stage. Yet some embryos with chomosomal abnormalities can develop beyond the critical first trimester of pregnancy and, while those with supernumary chromosomes in their hyperdiploid cells will be spontaneously aborted, a small fraction of fetuses with an extra chromosome continues to grow to term and will be delivered as a liveborn baby. While minor clinical symptoms displayed by children with trisomies are manageable for many parents, the burden of caring for a child with numerical chromosome abnormalities can be overwhelming to partners or individual families. It also poses a significant financial burden to the society and poses ethical dilemma. In this communication, we will review the progress that has been made in the development of molecular techniques to test individual fetal cells for chromosomal imbalances. We will focus our discussion on the direct visualization of chromosome-specific DNA sequences in live or fixed specimens using fluorescence in situ hybridization (FISH) and, more specifically, talk about the groundbreaking progress that in recent years has been achieved towards an improved diagnosis with novel, chromosome-specific DNA probes.
Fluorescence in situ hybridization (FISH)↗
Probing DNA clamps with single-molecule force spectroscopy
Detailed mechanisms of DNA clamps in prokaryotic and eukaryotic systems were investigated by probing their mechanics with single-molecule force spectroscopy. Specifically, the mechanical forces required for the Escherichia coli and Saccharomyces cerevisiae clamp opening were measured at the single-molecule level by optical tweezers. Steered molecular dynamics simulations further examined the forces involved in DNA clamp opening from the perspective of the interface binding energies associated with the clamp opening processes. In combination with additional molecular dynamics simulations, we identified the contact networks between the clamp subunits that contribute significantly to the interface stability of the S.cerevisiae and E. coli clamps. These studies provide a vivid picture of the mechanics and energy landscape of clamp opening and reveal how the prokaryotic and eukaryotic clamps function through different mechanisms.
59 BASIC BIOLOGICAL SCIENCES↗
Probing DNA structural heterogeneity by identifying conformational subensembles of a bicovalently bound cyanine dye
DNA is a re-configurable, biological information-storage unit, and much remains to be learned about its heterogeneous structural dynamics. For example, while it is known that molecular dyes templated onto DNA exhibit increased photostability, the mechanism by which the structural dynamics of DNA affect the dye photophysics remains unknown. Here, we use femtosecond, two-dimensional electronic spectroscopy measurements of a cyanine dye, Cy5, to probe local conformations in samples of single-stranded DNA (ssDNA–Cy5), double-stranded DNA (dsDNA–Cy5), and Holliday junction DNA (HJ–DNA–Cy5). A line shape analysis of the 2D spectra reveals a strong excitation–emission correlation present in only the dsDNA–Cy5 complex, which is a signature of inhomogeneous broadening. Molecular dynamics simulations support the conclusion that this inhomogeneous broadening arises from a nearly degenerate conformer found only in the dsDNA–Cy5 complex. These insights will support future studies on DNA’s structural heterogeneity.
77 NANOSCIENCE AND NANOTECHNOLOGY↗
Quantum dot-based microfluidic biosensor for cancer detection
We report results of the studies relating to fabrication of an impedimetric microfluidic–based nucleic acid sensor for quantification of DNA sequences specific to chronic myelogenous leukemia (CML). The sensor chip is prepared by patterning an indium–tin–oxide (ITO) coated glass substrate via wet chemical etching method followed by sealing with polydimethylsiloxane (PDMS) microchannel for fluid control. The fabricated microfluidic chip comprising of a patterned ITO substrate is modified by depositing cadmium selenide quantum dots (QCdSe) via Langmuir–Blodgett technique. Further, the QCdSe surface has been functionalized with specific DNA probe for CML detection. The probe DNA functionalized QCdSe integrated miniaturized system has been used to monitor target complementary DNA concentration by measuring the interfacial charge transfer resistance via hybridization. The presence of complementary DNA in buffer solution significantly results in decreased electro-conductivity of the interface due to presence of a charge barrier for transport of the redox probe ions. The microfluidic DNA biosensor exhibits improved linearity in the concentration range of 10{sup −15} M to 10{sup −11} M.
60 APPLIED LIFE SCIENCES↗
Robust, versatile DNA FISH probes for chromosome-specific repeats in Caenorhabditis elegans and Pristionchus pacificus
Repetitive DNA sequences are useful targets for chromosomal fluorescence in situ hybridization. We analyzed recent genome assemblies of Caenorhabditis elegans and Pristionchus pacificus to identify tandem repeats with a unique genomic localization. Based on these findings, we designed and validated sets of oligonucleotide probes for each species targeting at least 1 locus per chromosome. These probes yielded reliable fluorescent signals in different tissues and can easily be combined with the immunolocalization of cellular proteins. Synthesis and labeling of these probes are highly cost-effective and require no hands-on labor. The methods presented here can be easily applied in other model and nonmodel organisms with a sequenced genome.
59 BASIC BIOLOGICAL SCIENCES↗
Analytical Techniques Used to Detect DNA Binding Modes of Ruthenium(II) Complexes with Extended Phenanthroline Ring
This review describes the analytical techniques used to detect DNA-probes such as Ru(II) complexes with hetero cyclic imidazo phenanthroline (IP) ligands. Studies on drug-DNA interactions are useful biochemical techniques for visualization of DNA both in vitro and in vivo. The interactions of small molecules that binds to DNA are mainly classified into two major classes, one involving covalent binding and another non-covalent binding. Covalent binding in DNA can be irreversible and may leads to inhibition of all DNA processes which subsequently leads to cell death. Usually, covalent interactions leads to permanent changes in the structure of nucleic acids. The non-covalent interaction of molecules with DNA can be due to electrostatic interaction, intercalation and groove binding. These interactions of DNA probes can be explored by various spectroscopic techniques viz. UV–visible, emission, emission quenching spectroscopy, viscosity and thermal denaturation measurements.
74 ATOMIC AND MOLECULAR PHYSICS↗
Probing DNA-protein interactions using single-molecule diffusivity contrast
Not Available
Wilson, Hugh↗
Data Analysis for DNA Stable Isotope Probing Experiments Using Multiple Window High-Resolution SIP
DNA stable isotope probing (DNA-SIP) allows for the identification of microbes that assimilate isotopically labeled substrates into DNA. Here we describe the analysis of sequencing data using the multiple window high-resolution DNA-SIP method (MW-HR-SIP). MW-HR-SIP has improved accuracy over other methods and is easily implemented on the statistical platform R. Here, we also discuss key experimental parameters to consider when designing DNA-SIP experiments and how these parameters affect accuracy of analysis.
59 BASIC BIOLOGICAL SCIENCES↗
Single molecule insights into interfacial molecular recognition for model electrochemical DNA biosensors
Electrochemical sensors that use surface-immobilized DNA to bind analytes and transduce the binding into electrochemical signals, have the potential for rapid, specific, and sensitive detection of bioanalytes via a compact and portable platform. However, accessing the structure of these surfaces/interfaces at the relevant spatial scale (< 10 nm), which determines the interfacial interactions and ultimately sensing performance, remains an unsolved challenge. Here, we review studies that have used high resolution atomic force microscope imaging and spatial statistical analysis tools to understand crowding interactions between thiolated DNA probes immobilized on gold electrodes and how such interactions impact target binding. We also review related studies that attempt to control the nanoscale spatial arrangement of the immobilized recognition elements to optimize sensing performance. Furthermore, these efforts have led to new advances in understanding of the structure-function relationships of DNA-based electrochemical biosensors to move the field toward rational engineering of these biosensing interfaces.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DNA binding of the p21 repressor ZBTB2 is inhibited by cytosine hydroxymethylation
Highlights: • 5-hmC epigenetic modification is measurable in HeLa, SH-SY5Y and UT7-MPL cell lines. • ZBTB2 binds to DNA probes containing 5-mC but not to sequences containing 5-hmC. • This differential binding is verified with DNA sequences involved in p21 regulation. - Abstract: Recent studies have demonstrated that the modified base 5-hydroxymethylcytosine (5-hmC) is detectable at various rates in DNA extracted from human tissues. This oxidative product of 5-methylcytosine (5-mC) constitutes a new and important actor of epigenetic mechanisms. We designed a DNA pull down assay to trap and identify nuclear proteins bound to 5-hmC and/or 5-mC. We applied this strategy to three cancerous cell lines (HeLa, SH-SY5Y and UT7-MPL) in which we also measured 5-mC and 5-hmC levels by HPLC-MS/MS. We found that the putative oncoprotein Zinc finger and BTB domain-containing protein 2 (ZBTB2) is associated with methylated DNA sequences and that this interaction is inhibited by the presence of 5-hmC replacing 5-mC. As published data mention ZBTB2 recognition of p21 regulating sequences, we verified that this sequence specific binding was also alleviated by 5-hmC. ZBTB2 being considered as a multifunctional cell proliferation activator, notably through p21 repression, this work points out new epigenetic processes potentially involved in carcinogenesis.
60 APPLIED LIFE SCIENCES↗
Full Karyotype Interphase Cell Analysis
Aneuploidy seems to play not only a decisive role in embryonal development but also in tumorigenesis where chromosomal and genomic instability reflect a universal feature of malignant tumors. The cost of whole genome sequencing has fallen significantly, but it is still prohibitive for many institutions and clinical settings. No applied, cost-effective, and efficient technique has been introduced yet aiming at research to assess the ploidy status of all 24 different human chromosomes in interphases simultaneously, especially in single cells. Here, we present the selection of human probe DNA and a technique using multistep fluorescence in situ hybridization (FISH) employing four sets of six labeled FISH probes able to delineate all 24 human chromosomes in interphase cells. This full karyotype analysis approach will provide additional diagnostic potential for single cell analysis. The use of spectral imaging (SIm) has enabled the use of up to eight different fluorochrome labels simultaneously. Thus, scoring can be easily assessed by visual inspection, because SIm permits computer-assigned and distinguishable pseudo-colors to each probe during image processing. This enables full karyotype analysis by FISH of single-cell interphase nuclei.
Baumgartner, Adi↗
CNT-Based Label-Free Electrochemical Sensing of Native DNA with Allele Single Nucleotide Polymorphism
A label-free high-performance electrochemical sensing of single nucleotide polymorphism in native genomic DNA samples from cancer tissues, in oncogene-specific complementary DNA and in amplicons has been carried out on bundles of few-walled carboxylated carbon nanotubes, surface of which was coated with electron-dense layer of probe DNA. High selectivity and sensitivity of the novel DNA-nanosensor are ensured by the following features. Firstly, a complementary-hybridization signal is enhanced by resonance between plasmons in carbon nanotubes and dipole oscillations of hydrated nucleic acids located in near-electrode Helmholtz double layer of the impedance sensor. Secondly, a decoration of carbon nanotubes by adhered metal atoms in a high-spin state leads to Fermi level stabilization for the carbon nanotube bundles.
77 NANOSCIENCE AND NANOTECHNOLOGY↗
Electrochemical DNA biosensor for detection of porcine oligonucleotides using ruthenium(II) complex as intercalator label redox
A DNA biosensor detection of oligonucleotides via the interactions of porcine DNA with redox active complex based on the electrochemical transduction is described. A ruthenium(II) complex, [Ru(bpy){sub 2}(PIP)]{sup 2+}, (bpy = 2,2′bipyridine, PIP = 2-phenylimidazo[4,5-f[[1,10-phenanthroline]) as DNA label has been synthesized and characterized by 1H NMR and mass spectra. The study was carried out by covalent bonding immobilization of porcine aminated DNA probes sequences on screen printed electrode (SPE) modified with succinimide-acrylic microspheres and [Ru(bpy){sub 2}(PIP)]{sup 2+} was used as electrochemical redox intercalator label to detect DNA hybridization event. Electrochemical detection was performed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) over the potential range where the ruthenium (II) complex was active. The results indicate that the interaction of [Ru(bpy){sub 2}(PIP)]{sup 2+} with hybridization complementary DNA has higher response compared to single-stranded and mismatch complementary DNA.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Probe and method for DNA detection
A hybridization probe containing two linear strands of DNA lights up upon hybridization to a target DNA using silver nanoclusters that have been templated onto one of the DNA strands. Hybridization induces proximity between the nanoclusters on one strand and an overhang on the other strand, which results in enhanced fluorescence emission from the nanoclusters.
59 BASIC BIOLOGICAL SCIENCES↗
Multiple-Targeted Graphene-based Nanocarrier for Intracellular Imaging of mRNAs
Simultaneous detection and imaging of multiple intracellular messenger RNA (mRNAs) hold great significant for early cancer diagnostics and preventive medicine development. Herein, we propose a multiple-targeted graphene oxide (GO) nanocarrier that can simultaneously detect and image different type mRNAs in living cells. First of all, in vitro detection of multiple targets have been realized successfully based on the multiple-targeted GO nanocarrier with linear relationship ranging from 3 nM to 200 nM, as well as sensitive detection limit of 1.84 nM for manganese superoxide dismutase (Mn-SOD) mRNA and 2.45 nM for β-actin mRNA. Additionally, this nanosensing platform composed of fluorescent labeled single strand DNA probes and GO nanocarrier can identify Mn-SOD mRNA and endogenous mRNA of β-actin in living cancer cells, showing rapid response, high specificity, nuclease stability, and good biocompatibility during the cell imaging. Thirdly, changes of the expression levels of mRNA in living cells before or after the drug treatment can be monitored successfully. By using multiple ssDNA as probes and GO nanocarrier as the cellular delivery cargo, the proposed simultaneous multiple-targeted sensing platform will be of great potential as a powerful tool for intracellular trafficking process from basic research to clinical diagnosis.
59 BASIC BIOLOGICAL SCIENCES↗