Electron microscopic visualization of complementary labeled DNA with platinum-containing guanine derivative.

The object of the present report is to provide a method for a visualization of DNA in TEM by complementary labeling of cytosine with guanine derivative, which contains platinum as contrast-enhanced heavy element. The stretched single-chain DNA was obtained by modifying double-stranded DNA. The labeling method comprises the following steps: (i) stretching and adsorption of DNA on the support film of an electron microscope grid (the hydrophobic carbon film holding negative charged DNA); (ii) complementary labeling of the cytosine bases from the stretched single-stranded DNA pieces on the support film with platinum containing guanine derivative to form base-specific hydrogen bond; and (iii) producing a magnified image of the base-specific labeled DNA. Stretched single-stranded DNA on a support film is obtained by a rapid elongation of DNA pieces on the surface between air and aqueous buffer solution. The attached platinum-containing guanine derivative serves as a high-dense marker and it can be discriminated from the surrounding background of support carbon film and visualized by use of conventional TEM observation at 100 kV accelerated voltage. This method allows examination of specific nucleic macromolecules through atom-by-atom analysis and it is promising way toward future DNA-sequencing or molecular diagnostics of nucleic acids by electron microscopic observation.

Anisotropic nanoparticle complementarity in DNA-mediated co-crystallization.

Whether two species will co-crystallize depends on the chemical, physical and structural complementarity of the interacting components. Here, by using DNA as a surface ligand, we selectively co-crystallize mixtures of two different anisotropic nanoparticles and systematically investigate the effects of nanoparticle size and shape complementarity on the resultant crystal symmetry, microstrain, and effective 'DNA bond' length and strength. We then use these results to understand a more complicated system where both size and shape complementarity change, and where one nanoparticle can participate in multiple types of directional interactions. Our findings offer improved control of non-spherical nanoparticles as building blocks for the assembly of sophisticated macroscopic materials, and provide a framework to understand complementarity and directional interactions in DNA-mediated nanoparticle crystallization.

Visualising single molecules of HIV-1 and miRNA nucleic acids.

BACKGROUND: The scarcity of certain nucleic acid species and the small size of target sequences such as miRNA, impose a significant barrier to subcellular visualization and present a major challenge to cell biologists. Here, we offer a generic and highly sensitive visualization approach (oligo fluorescent in situ hybridization, O-FISH) that can be used to detect such nucleic acids using a single-oligonucleotide probe of 19-26 nucleotides in length. RESULTS: We used O-FISH to visualize miR146a in human and avian cells. Furthermore, we reveal the sensitivity of O-FISH detection by using a HIV-1 model system to show that as little as 1-2 copies of nucleic acids can be detected in a single cell. We were able to discern newly synthesized viral cDNA and, moreover, observed that certain HIV RNA sequences are only transiently available for O-FISH detection. CONCLUSIONS: Taken together, these results suggest that the O-FISH method can potentially be used for in situ probing of, as few as, 1-2 copies of nucleic acid and, additionally, to visualize small RNA such as miRNA. We further propose that the O-FISH method could be extended to understand viral function by probing newly transcribed viral intermediates; and discern the localisation of nucleic acids of interest. Additionally, interrogating the conformation and structure of a particular nucleic acid in situ might also be possible, based on the accessibility of a target sequence.

Identifying individual DNA species in a complex mixture by precisely measuring the spacing between nicking restriction enzymes with atomic force microscope.

We discuss a novel atomic force microscope-based method for identifying individual short DNA molecules (<5000 bp) within a complex mixture by measuring the intra-molecular spacing of a few sequence-specific topographical labels in each molecule. Using this method, we accurately determined the relative abundance of individual DNA species in a 15-species mixture, with fewer than 100 copies per species sampled. To assess the scalability of our approach, we conducted a computer simulation, with realistic parameters, of the hypothetical problem of detecting abundance changes in individual gene transcripts between two single-cell human messenger RNA samples, each containing roughly 9000 species. We found that this approach can distinguish transcript species abundance changes accurately in most cases, including transcript isoforms which would be challenging to quantitate with traditional methods. Given its sensitivity and procedural simplicity, our approach could be used to identify transcript-derived complementary DNAs, where it would have substantial technical and practical advantages versus established techniques in situations where sample material is scarce.

Induction of intracellular tau aggregation is promoted by α-synuclein seeds and provides novel insights into the hyperphosphorylation of tau.

Intracytoplasmic proteinaceous inclusions, primarily composed of tau or α-synuclein (α-syn), are predominant pathological features of Alzheimer's disease (AD) and Parkinson's disease (PD), respectively. However, the coexistence of these pathological aggregates is identified in many neurodegenerative disorders, including spectrum disorders of AD and PD. Whereas α-syn can spontaneously polymerize into amyloidogenic fibrils, in vitro, tau polymerization requires an inducing agent. The current study presents a human-derived cellular model, in which recombinant, preformed α-syn fibrils cross-seed intracellular tau to promote the formation of neurofibrillary tangle-like aggregates. These aggregates were hyperphosphorylated, Triton insoluble, and thioflavin-S positive, either comingling with endogenously expressed α-syn aggregates or induced by only exogenously applied recombinant α-syn fibrils. Furthermore, filamentous, amyloidogenic tau took over the cellular soma, displacing the nucleus and isolating or displacing organelles, likely preventing cellular function. Although a significant proportion of wild-type tau formed these cellular inclusions, the P301L mutation in tau increased aggregation propensity resulting from α-syn seeds to over 50% of total tau protein. The role of phosphorylation on the development of these tau aggregates was investigated by coexpressing glycogen synthase kinase 3 ß or microtubule-associated protein/microtubule affinity-regulating kinase 2. Expression of either kinase inhibited the formation of α-syn-induced tau aggregates. Analyses of phosphorylation sites suggest that multiple complex factors may be associated with this effect and that Triton-soluble versus Triton-insoluble tau may be independently targeted by kinases. The current work not only provides an exceptional cellular model of tau pathology, but also examines α-syn-induced tau inclusion formation and provides novel insights into hyperphosphorylation observed in disease.

Noise reduction of cDNA microarray images using complex wavelets.

Noise reduction is an essential step of cDNA microarray image analysis for obtaining better-quality gene expression measurements. Wavelet-based denoising methods have shown significant success in traditional image processing. The complex wavelet transform (CWT) is preferred to the classical discrete wavelet transform for denoising of microarray images due to its improved directional selectivity for better representation of the circular edges of spots and near shift-invariance property. Existing CWT-based denoising methods are not efficient for microarray image processing because they fail to take into account the signal as well as noise correlations that exist between red and green channel images. In this paper, two bivariate estimators are developed for the CWT-based denoising of microarray images using the standard maximum a posteriori and linear minimum mean squared error estimation criteria. The proposed denoising methods are capable of taking into account both the interchannel signal and noise correlations. Significance of the proposed denoising methods is assessed by examining the effect of noise reduction on the estimation of the log-intensity ratio. Extensive experimentations are carried out to show that the proposed methods provide better noise reduction of microarray images leading to more accurate estimation of the log-intensity ratios as compared to the other CWT-based denoising methods.

Expression profiling of fungal genes during arbuscular mycorrhiza symbiosis establishment using direct fluorescent in situ RT-PCR.

Expression profiling of fungal genes in the arbuscular mycorrhiza (AM) symbiosis has been based on studies of RNA extracted from fungal tissue or mycorrhizal roots, giving only a general picture of overall transcript levels in the targeted tissues. Information about the spatial distribution of transcripts within AM fungal structures during different developmental stages is essential to a better understanding of fungal activity in symbiotic interactions with host roots and to determine molecular events involved in establishment and functioning of the AM symbiosis. The obligate biotrophic nature of AM fungi is a challenge for developing new molecular methods to identify and localize their activity in situ. The direct fluorescent in situ (DIFIS) RT-PCR procedure described here represents a novel tool for spatial mapping of AM fungal gene expression simultaneously prior to root penetration, within fungal tissues in the host root and in the extraradical stage of fungal development.In order to enhance detection sensitivity of the in situ RT-PCR technique and enable localization of low abundance mRNA, we have adopted direct fluorescent labeling of primers for the amplification step to overcome the problem of low detection associated with digoxigenin or biotin-labeled primers and to avoid the multiplicity of steps associated with immunological detection. Signal detection has also been greatly improved by eliminating autofluorescence of AM fungal and root tissues using confocal microscopy.

Electrochemical detection of a breast cancer susceptible gene using cDNA immobilized chitosan-co-polyaniline electrode.

An electrochemical breast cancer biosensor based on a chitosan-co-polyaniline (CHIT-co-PANI) copolymer coated onto indium-tin-oxide (ITO) was fabricated by immobilizing the complementary DNA (cDNA) probe (42 bases long) associated with the breast cancer susceptible gene BRCA1. Both the CHIT-co-PANI/ITO and the cDNA/CHIT-co-PANI/ITO electrodes were characterized with Fourier transform infrared (FTIR) spectroscopy, atomic force microscopy (AFM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). For the cDNA/CHIT-co-PANI/ITO electrode, the amperometric current decreased linearly with an increasing logarithm of molar concentration of the single-stranded target DNA (ssDNA) within the range of 0.05-25fmol. The bioelectrode exhibited a sensitivity of 2.104 microA/fmol with a response time of 16s. The cDNA/CHIT-co-PANI/ITO electrode had a shelf life of about six months, even when stored at room temperature.

DNA-drug interaction measurements using surface plasmon resonance.

The interactions of the drugs 2,7-bis[(diethylamino)-ethoxy]-fluoren-9-one dihydrochloride (Tilorone), 2,7-bis[(dipropylamino)-acetamido]-fluoren-9-one dihydrochloride (FA-2), 2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H-benzimidazole trihydrochloride (Hoechst 33258), and hematoporphyrin IX derivative (HPD) with synthetic self-complementary DNA (36-b.p.; 5'-biotin-spacer-[d(CGCTATATAGCG)]3-3') were studied by SPR (Surface Plasmon Resonance). Monolayers of biotinylated DNA were immobilized on a streptavidin-dextran-gold triple-layer. Small portions of the drugs (approximately 30 pmol/ml) were injected in continuous flow. The mass corresponded to the amount of the bound molecules. Injections of 50 mM sodium hydroxide pulses separated the DNA double strands, releasing the effector molecules. Subsequent treatments with the effectors gave reproducible results. The maximum interaction between drug and DNA was observed in the case of Tilorone. 41 molecules could bind to the 36-b.p. DNA duplex. To investigate the microscopic behavior in condensed nucleic acid phases, SFM (Scanning Force Microscopy)-imaging and polarizing microscopic observations of DNA-effector complexes were carried out. Supplementary UV-absorption thermal denaturation curves of DNA with the above-mentioned effectors in dilute solutions were measured. As an additional aid to understand the geometries of DNA-drug interactions, computer simulations were performed and compared with the experimental data.

Brain cytochrome oxidase subunit complementary DNAs: isolation, subcloning, sequencing, light and electron microscopic in situ hybridization of transcripts, and regulation by neuronal activity.

The goal of the present study was to isolate, for the first time, cytochrome oxidase subunit genes from murine brain complementary DNA library and to characterize the expression of these genes from mitochondrial and nuclear sources at both light and electron microscopic levels. Brain subunit III (mitochondrial) shared 100% identity with that of murine L cells. Subunit VIa (nuclear) was known to have tissue-specific isoforms in other species: the ubiquitous liver isoform and the heart/muscle isoform. Our brain subunit VIa shared 93% homology with that of the rat liver and 100% identity with the recently reported murine liver isoform, which is only 62% identical to that of the rat heart isoform. In situ hybridization with riboprobes revealed messenger RNA labelling that was similar, though not identical, to that of cytochrome oxidase histochemistry. Monocular enucleation in adult mice induced a significant down-regulation of both subunit messages in the contralateral lateral geniculate nucleus. However, the decrease in subunit III messenger RNAs surpassed that of subunit VIa at all time periods examined, suggesting that mitochondrial gene expression is more tightly regulated by neuronal activity than that of nuclear ones. At the electron microscopic level, subunit III messenger RNA was localized to the mitochondrial compartment in both cell bodies and processes, while that of nuclear-encoded subunit VIa was present exclusively in the extramitochondrial compartment of somata and not of dendrites or axons. Surprisingly, the message was primarily associated with the rough endoplasmic reticulum, suggesting a novel pathway for its synthesis and trafficking. Our results indicate that the unique properties of neurons impose special requirements for subunits of a single mitochondrial enzyme with dual genomic origins. At sites of high energy demands (such as postsynaptic dendrites and some axon terminals), mitochondrial-encoded cytochrome oxidase subunits can be locally transcribed and translated, and they provide the framework for the subsequent importation and incorporation of nuclear-encoded subunits, which are strictly synthesized in the cell bodies. Dynamic local energy needs are met when subunits from the two genomic sources are assembled to form functional holoenzymes.

Exon mapping by fiber-FISH or LR-PCR.

In this study we systematically assessed the sensitivity limits of fiber-FISH in model experiments. Exonic fragments and cDNAs with exon sizes of >/=200 bp could be mapped on their cognate cosmid. This positional fiber-FISH mapping was validated by long-range PCR. It is expected that these two independent mapping approaches will help to refine current available gene maps and show their applicability in fine mapping of sequence-tagged sites or expressed sequence tags. Also, they will be useful in resolving gene structures by mapping exon and intron locations.