Fluorescence-labeled DNA Research Articles

Superresolution imaging of single DNA molecules using stochastic photoblinking of minor groove and intercalating dyes

As proof-of-principle for generating superresolution structural information from DNA we applied a method of localization microscopy utilizing photoblinking comparing intercalating dye YOYO-1 against minor groove binding dye SYTO-13, using a bespoke multicolor single-molecule fluorescence microscope. We used a full-length ∼49kbp λ DNA construct possessing oligo inserts at either terminus allowing conjugation of digoxigenin and biotin at opposite ends for tethering to a glass coverslip surface and paramagnetic microsphere respectively. We observed stochastic DNA-bound dye photoactivity consistent with dye photoblinking as opposed to binding/unbinding events, evidenced through both discrete simulations and continuum kinetics analysis. We analyzed dye photoblinking images of immobilized DNA molecules using superresolution reconstruction software from two existing packages, rainSTORM and QuickPALM, and compared the results against our own novel home-written software called ADEMS code. ADEMS code generated lateral localization precision values of 30–40nm and 60–70nm for YOYO-1 and SYTO-13 respectively at video-rate sampling, similar to rainSTORM, running more slowly than rainSTORM and QuickPALM algorithms but having a complementary capability over both in generating automated centroid distribution and cluster analyses. Our imaging system allows us to observe dynamic topological changes to single molecules of DNA in real-time, such as rapid molecular snapping events. This will facilitate visualization of fluorescently-labeled DNA molecules conjugated to a magnetic bead in future experiments involving newly developed magneto-optical tweezers combined with superresolution microscopy.

Tension-Dependent Stability of Torsionally Constrained DNA: Melting Precedes Overwinding

Key genomic processes such as replication and transcription require localized unwinding and base-pair melting of the double-helix. In vivo, however, DNA is often torsionally constrained, presenting a topological barrier to unwinding. While it is known that unwinding can proceed through buckling of such DNA (resulting in plectonemes), this may only occur at low forces (typically below 1 pN). Here, we ask: how can torsionally constrained DNA unwind at higher forces without changing the overall twist?Using a combination of optical tweezers and fluorescence microscopy we map the local changes in twist and base-pair integrity of torsionally constrained DNA as a function of tension and ionic strength. By correlating the second derivatives of force-extension curves of torsionally constrained DNA to the binding of a fluorescently-labeled single-stranded DNA binding protein (Replication Protein A), we identify two tension-dependent structural transitions. In agreement with a previous proposal, we show that at high tension (above 115 pN), unwinding is accompanied by the formation of overwound DNA (termed P-DNA) in a largely cooperative mechanism. Strikingly, we also reveal that for intermediate tensions (60 to 115 pN), localized base-pair melting can occur without changes in DNA twist. This mechanism, which is significantly less cooperative, is referred to here as ‘pre-melting’. We demonstrate that pre-melting is favored by AT-rich sequences and low ionic strength. In contrast, P-DNA is largely stabilized by higher salt concentrations, while also having a preference for AT-rich domains. This supports the hypothesis that P-DNA consists of tightly entwined backbones with exposed bases.These findings provide a new understanding of the interplay between DNA twist, extension and sequence. Moreover, since DNA unwinding can be induced through either tension or applied torque, this work may have strong implications for the metabolism of torsionally constrained DNA in vivo.

Fluorescent labelling of DNA on superparamagnetic nanoparticles by a perylene bisimide derivative for cell imaging

N,N'-Bis[tris-(2-aminoethyl) amine]-3,4,9,10-perylenetetracarboxylic diimide (PBI-TRIS), nonfluorescent dye was used to fluorescent labelling of DNA. For this aim, (3-aminopropyl) triethoxysilane (APTS) modified superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized to provide a suitable surface for binding of DNA. Amine functionalized nanoparticles showed a high immobilization capacity (82.70%) at 25mg of nanoparticle concentration for Calf thymus DNA. Binding capacity of PBI-TRIS to DNA-SPION was also found as 1.93μM on 25mg of nanoparticles by using UV-vis spectroscopy. Binding of PBI-TRIS to DNA onto nanoparticles was also characterized by scanning electron microscopy and infrared spectroscopy. The confocal images of PBI-TRIS labelled DNA-SPION and breast cells were taken at 488 and 561.7nm of excitation wavelengths. Cell image was also compared with a commercial dye, DAPI at 403.7nm of excitation wavelength. Results showed that PBI-TRIS can be used for cell staining.

Simultaneous detection of ochratoxin A and fumonisin B1 in cereal samples using an aptamer-photonic crystal encoded suspension array.

A simple, new aptamer-photonic crystal encoded suspension array was designed to simultaneously quantify and qualify ochratoxin A(OTA) and fumonisin B1(FB1) in cereal samples. The aptamers of OTA and FB1 were immobilized on the surfaces of photonic crystals by chemical bonding. When the target mycotoxins appear in a sample, the fluorescence-labeled complementary DNA of the aptamer dissociates from their double DNA hybrid and results in an obvious decrease in fluorescence intensity of the microsphere. The difference value of fluorescent intensities for each kind of silica photonic crystal microsphere (SPCM) quantitatively conveys the concentration of mycotoxin, and the structure colors or reflectance peak positions of the SPCMs confirm the kind of mycotoxin detected. The reaction conditions including the immobilization method for aptamers, hybridization, and incubation conditions have been optimized. This developed method displayed a wide linear detection range (0.01-1 ng/mL for OTA and 0.001-1 ng/mL for FB1) and a low limit of detection (0.25 pg/mL for OTA and 0.16 pg/mL for FB1). The recovery rates in the spiked cereal samples ranged from 81.80% to 116.38% for OTA and 76.58%-114.79% for FB1. The positive detection results in the naturally contaminated cereal samples were in agreement with those of classic enzyme-linked immunosorbent assay (ELISA). This simple suspension array scheme displays a great application potential for the high throughput screen assay of mycotoxins.

Rapid Catch and Signal (RCS) Technology Platform: Multiplexed Three-Color, 30 s Microwave-Accelerated Metal-Enhanced Fluorescence DNA Assays

We present an innovative “Rapid Catch and Signal” (DNA-RCS) technology for the simultaneous highly selective detection of multiple specific DNA sequences in solution. The DNA-RCS technology combines advantages of microwave-accelerated DNA hybridization (Rapid Catch) with the metal-enhanced fluorescence (MEF) technology (Signal), to enable specific DNA’s to be detected at high sensitivity within seconds. Fluorescent DNA labels, which play the role of molecular sensor probes, show a strong response upon DNA hybridization, due to fluorophore coupling with nanoparticle plasmons at a short (10–30 nm) distance from the surface. We have also shown that the fluorophore sensor probes demonstrate high photostability due to close proximity to a SiF surface, which significantly increases the total stability and reliability of the assay. Applications of the DNA-RCS technology in the life sciences, its advantages and benefits as compared to other DNA detection schemes, such as PCR, are subsequently discussed.

Preparation and properties of clickable amino analogues of the duocarmycins: factors that affect the efficiency of their fluorescent labelling of DNA.

Herein we report the synthesis of three DNA-alkylating amino analogues of the duocarmycins that carry an alkyne functional group suitable for copper-catalysed click chemistry. The alkyne-containing substituents are connected via a side chain position which projects away from the minor groove, and have only a small effect on DNA alkylation and cytotoxicity. The efficiency of click reactions with fluorophore azides was studied using alkylated ctDNA by analysing the adenine adducts produced after thermal depurination. Click reactions "on DNA" were sensitive to steric effects (tether length to the alkyne) and, surprisingly, to the nature of the fluorophore azide. With the best combination of click partners and reagents, adducts could be detected in the nuclei of treated cells by microscopy or flow cytometry, provided that an appropriate detergent (Triton X-100 and not Tween 20) was used for permeabilisation. The method is sensitive enough to detect adducts at physiologically relevant concentrations, and could have application in the development of nitro analogues of the duocarmycins as hypoxia-activated anticancer prodrugs.

A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green

The increasing need for multiple-labeling of cells and whole organisms for fluorescence microscopy has led to the development of hundreds of fluorophores that either directly recognize target molecules or organelles, or are attached to antibodies or other molecular probes. DNA labeling is essential to study nuclear-chromosomal structure, as well as for gel staining, but also as a usual counterstain in immunofluorescence, FISH or cytometry. However, there are currently few reliable red to far-red-emitting DNA stains that can be used. We describe herein an extremely simple, inexpensive and robust method for DNA labeling of cells and electrophoretic gels using the very well-known histological stain methyl green (MG). MG used in very low concentrations at physiological pH proved to have relatively narrow excitation and emission spectra, with peaks at 633 and 677 nm, respectively, and a very high resistance to photobleaching. It can be used in combination with other common DNA stains or antibodies without any visible interference or bleed-through. In electrophoretic gels, MG also labeled DNA in a similar way to ethidium bromide, but, as expected, it did not label RNA. Moreover, we show here that MG fluorescence can be used as a stain for direct measuring of viability by both microscopy and flow cytometry, with full correlation to ethidium bromide staining. MG is thus a very convenient alternative to currently used red-emitting DNA stains.

Comparison of the Sequence-Dependent Fluorescence of the Cyanine Dyes Cy3, Cy5, DyLight DY547 and DyLight DY647 on Single-Stranded DNA

Cyanine dyes are commonly used for fluorescent labeling of DNA and RNA oligonucleotides in applications including qPCR, sequencing, fluorescence in situ hybridization, Förster resonance energy transfer, and labeling for microarray hybridization. Previous research has shown that the fluorescence efficiency of Cy3 and Cy5, covalently attached to the 5′ end of single-stranded DNA, is strongly sequence dependent. Here, we show that DY547 and DY647, two alternative cyanine dyes that are becoming widely used for nucleic acid labeling, have a similar pattern of sequence-dependence, with adjacent purines resulting in higher intensity and adjacent cytosines resulting in lower intensity. Investigated over the range of all 1024 possible DNA 5mers, the intensities of Cy3 and Cy5 drop by ∼50% and ∼65% with respect to their maxima, respectively, whereas the intensities of DY547 and DY647 fall by ∼45% and ∼40%, respectively. The reduced magnitude of change of the fluorescence intensity of the DyLight dyes, particularly of DY647 in comparison with Cy5, suggests that these dyes are less likely to introduce sequence-dependent bias into experiments based on fluorescent labeling of nucleic acids.

Fluorescence resonance energy transfer based aptasensor for the sensitive and selective detection of 17β-estradiol using a quantum dot-bioconjugate as a nano-bioprobe

A unique fluorescence resonance energy transfer (FRET)-based aptasensor was constructed for the determination of 17β-estradiol using a quantum dot (QD) bioconjugate as a nano-bioprobe and a high-affinity, high-specificity fluorescence-labeled anti-17β-estradiol aptamer as a bio-recognition molecule. Based on the principles of QD-FRET and competitive bioassays, a number of samples containing various 17β-estradiol concentrations and a given concentration of the fluorescence-labeled DNA aptamer were incubated with a certain concentration of the QD nano-bioprobe solution. A higher 17β-estradiol concentration led to fewer fluorescence-labelled DNA aptamers bound to the QD nano-bioprobe surface and thus a lower fluorescence signal. The compound 17β-estradiol was quantified over a concentration range of 0.82 nM to 20.5 nM with a detection limit of 0.22 nM. The high specificity and selectivity of the sensor were demonstrated by evaluating the response of the sensor toward a number of potentially interfering endocrine-disrupting compounds or other chemicals. This sensor was also sufficiently stable for practical applications. The performance of the FRET-based aptasensor with spiked water samples demonstrated good recovery, precision and accuracy, indicating that it had a low susceptibility to water matrix effects.

Intracellular Locations of Replication Proteins and the Origin of Replication during Chromosome Duplication in the Slowly Growing Human Pathogen Helicobacter pylori

We followed the position of the replication complex in the pathogenic bacterium Helicobacter pylori using antibodies raised against the single-stranded DNA binding protein (HpSSB) and the replicative helicase (HpDnaB). The position of the replication origin, oriC, was also localized in growing cells by fluorescence in situ hybridization (FISH) with fluorescence-labeled DNA sequences adjacent to the origin. The replisome assembled at oriC near one of the cell poles, and the two forks moved together toward the cell center as replication progressed in the growing cell. Termination and resolution of the forks occurred near midcell, on one side of the septal membrane. The duplicated copies of oriC did not separate until late in elongation, when the daughter chromosomes segregated into bilobed nucleoids, suggesting sister chromatid cohesion at or near the oriC region. Components of the replication machinery, viz., HpDnaB and HpDnaG (DNA primase), were found associated with the cell membrane. A model for the assembly and location of the H. pylori replication machinery during chromosomal duplication is presented.

Aptamer-Based Microspheres for Highly Sensitive Protein Detection Using Fluorescently-Labeled DNA Nanostructures

Many highly sensitive protein detection techniques have been developed and have played an important role in the analysis of proteins. Herein, we report a novel technique that can detect proteins sensitively and effectively using aptamer-based DNA nanostructures. Thrombin was used as a target protein and aptamer was used to capture fluorescent dye-labeled DNA nanobarcodes or thrombin on a microsphere. The captured DNA nanobarcodes were replaced by a thrombin and aptamer interaction. The detection ability of this approach was confirmed by flow cytometry with different concentrations of thrombin. Our detection method has great potential for rapid and simple protein detection with a variety of aptamers.

A Rapid and High-Throughput Quantitation Assay of the Nuclear Factor κB Activity Using Fluorescence Correlation Spectroscopy in the Setting of Clinical Laboratories

BackgroundTranscription factor nuclear factor-κB (NF-κB) plays a key role in the regulation of immune responses to inflammation. However, convenient assay systems to quantitate the NF-κB activity level in a timely manner are not available in the setting of clinical laboratories. Therefore, we developed a novel and high-throughput quantitative assay based on fluorescence correlation spectroscopy (FCS) to detect the NF-κB activity level in cellular nuclear extracts and evaluated the performance of this method. The basic principle of this assay is to calculate the binding fraction of NF-κB to fluorescent-labeled DNA probes, which contain NF-κB binding sites.MethodsNon-fluorescent competitive probes are employed to normalize the influence of the viscosity of the nuclear extracts between samples and to eliminate the influence of nonspecific binding of the fluorescent probes. To confirm accurate quantitation, human recombinant NF-κB p50 was mixed into U937 cell nuclear extracts, and the binding fraction of the fluorescent probes to NF-κB in the mixture was calculated for quantitation. To evaluate whether this method can be applied to measure the NF-κB activity in human lymphocytes, the NF-κB activity levels of systemic inflammatory response syndrome patients during perioperative periods were measured.ResultsThe percentage recovery was 88.9%. The coefficients of variation of the intra-assay were approximately 10%. NF-κB activity levels during the perioperative period can were successfully measured. The assay time for the FCS measurement was within 20 minutes.ConclusionsThis assay system can be used to quantitate NF-κB activity levels in a timely manner in the setting of hospital laboratories.

Rapid on-site/in-situ detection of heavy metal ions in environmental water using a structure-switching DNA optical biosensor

A structure-switching DNA optical biosensor for rapid on-site/in situ detection of heavy metal ions is reported. Mercury ions (Hg2+), highly toxic and ubiquitous pollutants, were selected as model target. In this system, fluorescence-labeled DNA containing T-T mismatch structure was introduced to bind with DNA probes immobilized onto the sensor surface. In the presence of Hg2+, some of the fluorescence-labeled DNAs bind with Hg2+ to form T-Hg2+-T complexes through the folding of themselves into a hairpin structure and dehybridization from the sensor surface, which leads to decrease in fluorescence signal. The total analysis time for a single sample was less than 10 min with detection limit of 1.2 nM. The rapid on-site/in situ determination of Hg2+ was readily performed in natural water. This sensing strategy can be extended in principle to other metal ions by substituting the T-Hg2+-T complexes with other specificity structures that selectively bind to other analytes.

Optimization and Application of a Custom Microarray for the Detection and Genotyping of E. coli O157:H7 in Fresh Meat Samples

DNA microarrays are promising high-throughput tools for multiple pathogen detection. Currently, the performance and cost of this platform has limited its broad application in identifying microbial contaminants in foods. In this study, an optimized custom DNA microarray with flexibility in design and content for foodborne pathogen detection was developed through the systematic evaluation of spotting buffers, probe lengths, scanning conditions, and nucleic acid amplification and labeling strategies. Briefly, by robotic contact printing, a spotting solution of 50 % dimethylsulfoxide produced uniform and high-quality spots on UltraGAPS glass slides coated with aminopropyl silane. The use of 60 % photomultiplier tube gain in scanning ∼70-mer oligonucleotide probes resulted in strong signals and low background. For sample preparation, multiplex PCR amplification coupled with fluorescent labeling of DNA using the Klenow fragment and random hexamers achieved higher specificity than whole genome random amplification. To minimize the cost of the assay, the quantities of probes, Klenow fragment, and Cy5 were substantially reduced in each assay without noticeably affecting the detection efficiency. Applying the optimized microarray assay to 26 fresh meat samples, three different isolates of Escherichia coli O157:H7 were found in four individual packages, demonstrating that the assay has a great potential for identifying and genotyping multiple pathogens in a real food system.

The (TTTA) n polymorphism in intron 4 of CYP19 and the polycystic ovary syndrome risk in a Chinese population

Polycystic ovary syndrome (PCOS) is a heterogeneous disease with a strong genetic origin, but the specific determinants are still unknown. The aim of this study was to investigate the association between the (TTTA) n polymorphism in intron 4 of CYP19 and the PCOS risk in a Chinese population. We performed a case-control study which involved 222 PCOS patients and 281 controls. The fluorescent-labeled target DNA fragments containing the (TTTA)n short tandem repeats were obtained by PCR, thereafter genotyped via capillary electrophoresis. Representative alleles were directly sequenced to confirm their repeat numbers. Genotype analysis revealed seven different alleles including 7-3(∆)-, 7-, 8-, 10-, 11-, 12- and 13-TTTA-repeats. The most common allele in a Chinese population is (TTTA) 11 in our study (0.354 for PCOS and 0.390 for controls). PCOS patients showed a higher frequency of short alleles compared with controls (0.47 vs. 0.41, OR=1.245, 95% CI 0.97-1.60). The overall allelic distributions of this polymorphism did not show any significant differences between PCOS patients and the control group. No statistical differences were found in the clinical parameters or serum steroid hormone levels among PCOS patients with different genotypes. In conclusion, PCOS patients had a higher frequency of short alleles, albeit this might not strongly affect the risk of PCOS.

Sentinel lymph node imaging by a fluorescently labeled DNA tetrahedron

Sentinel lymph nodes (SLNs) are the first lymph nodes which cancer cells reach after traveling through lymphatic vessels from the primary tumor. Evaluating the nodal status is crucial in accurate staging of human cancers and accordingly determines prognosis and the most appropriate treatment. The commonly used methods for SLN identification in clinics are based on employment of a colloid of radionuclide or injection of a small dye. Although these methods have certainly contributed to improve surgical practice, new imaging materials are still required to overcome drawbacks of the techniques such as inconvenience of handling radioactive materials and short retention time of small dyes in SLNs. Here, we prepare a fluorescence-labeled DNA tetrahedron and perform SLN imaging by using the DNA nanoconstruct. With a successful identification of SLNs by the DNA nanoconstruct, we suggest that DNA tetrahedron hold great promises for clinical applications.

Determination of micro‐RNA in cardiomyoblast cells using CE with LIF detection

Micro-RNAs (miRNAs) are small, endogenous, singlestranded, and noncoding RNAs. The miRNAs have been found to perform important functions in many cellular processes, such as development, proliferation, differentiation, and apoptosis. Circulating miRNAs have been proposed as emerging biomarkers in diseases such as cancer, diabetes, and cardiovascular disease including acute myocardial infarction (AMI). In this study, we developed CE with LIF (CE-LIF) using fluorescence-labeled DNA probe for determination of low abundance miRNA in cell extracts. The target miRNA is miRNA-499, a biomarker candidate of AMI with low abundance in biological samples. In order to measure the trace level of miRNA, we optimized the hybridization conditions such as hybridization time, temperature, and buffer solution. The highest fluorescence intensity of the hybridized miRNA-499 was found when hybridization was conducted at 40°C in 50 mM Tris-acetate (pH 8.0) buffer containing 50 mM NaCl, and 10 mM EDTA for 15 min. The hybridized miRNA-499 was detected in cultured H9c2 cardiomyoblast cells and the analysis of miRNA-499 was completed within 1 h using CE-LIF. These results showed the potential of CE for fast, specific, and sensitive high-throughput analysis of low-abundance miRNAs in cell extracts, biofluids, and tissues.

Caulobacter chromosome in vivo configuration matches model predictions for a supercoiled polymer in a cell-like confinement

We measured the distance between fluorescent-labeled DNA loci of various interloci contour lengths in Caulobacter crescentus swarmer cells to determine the in vivo configuration of the chromosome. For DNA segments less than about 300 kb, the mean interloci distances, <r>, scale as n(0.22), where n is the contour length, and cell-to-cell distribution of the interloci distance r is a universal function of r/n(0.22) with broad cell-to-cell variability. For DNA segments greater than about 300 kb, the mean interloci distances scale as n, in agreement with previous observations. The 0.22 value of the scaling exponent for short DNA segments is consistent with theoretical predictions for a branched DNA polymer structure. Predictions from Brownian dynamics simulations of the packing of supercoiled DNA polymers in an elongated cell-like confinement are also consistent with a branched DNA structure, and simulated interloci distance distributions predict that confinement leads to "freezing" of the supercoiled configuration. Lateral positions of labeled loci at comparable positions along the length of the cell are strongly correlated when the longitudinal locus positions differ by <0.16 μm. We conclude that the chromosome structure is supercoiled locally and elongated at large length scales and that substantial cell-to-cell variability in the interloci distances indicates that in vivo crowding prevents the chromosome from reaching an equilibrium arrangement. We suggest that the force causing rapid transport of loci remote from the parS centromere to the distal cell pole may arise from the release at the polar region of potential energy within the supercoiled DNA.

Gold nanoparticles and fluorescently-labelled DNA as a platform for biological sensing

In the past decade gold nanoparticle-nucleic acid conjugates became progressively important for biomedical applications. Fluorophores attached to nucleic acid-gold nanoparticle conjugates have opened up a new era of biological sensing. The most promising advancement in this field was the invention of the so-called 'nano-flare' systems. These systems are capable of detecting specific endocellular targets such as mRNAs, microRNAs or small molecules in real time. In this minireview, we discuss the current progress in the field of DNA-nanoparticles as sensors, their properties, stability, cellular uptake and cytotoxicity.

Identification of HIV-1 Inhibitors Targeting the Nucleocapsid Protein

The HIV-1 nucleocapsid (NC) is a RNA/DNA binding protein encoded within the Gag polyprotein, which is critical for the selection and chaperoning of viral genomic RNA during virion assembly. RNA/DNA binding occurs through a highly conserved zinc-knuckle motif present in NC. Given the necessity of NC-viral RNA/DNA interaction for viral replication, identification of compounds that disrupt the NC-RNA/DNA interaction may have value as an antiviral strategy. To identify small molecules that disrupt NC-viral RNA/DNA binding, a high-throughput fluorescence polarization assay was developed and a library of 14,400 diverse, druglike compounds was screened. Compounds that disrupted NC binding to a fluorescence-labeled DNA tracer were next evaluated by differential scanning fluorimetry to identify compounds that must bind to NC or Gag to impart their effects. Two compounds were identified that inhibited NC-DNA interaction, specifically bound NC with nanomolar affinity, and showed modest anti-HIV-1 activity in ex vivo cell assays.