Customized optical mapping by CRISPR-Cas9 mediated DNA labeling with multiple sgRNAs.

Whole-genome mapping technologies have been developed as a complementary tool to provide scaffolds for genome assembly and structural variation analysis (1,2). We recently introduced a novel DNA labeling strategy based on a CRISPR-Cas9 genome editing system, which can target any 20bp sequences. The labeling strategy is specifically useful in targeting repetitive sequences, and sequences not accessible to other labeling methods. In this report, we present customized mapping strategies that extend the applications of CRISPR-Cas9 DNA labeling. We first design a CRISPR-Cas9 labeling strategy to interrogate and differentiate the single allele differences in NGG protospacer adjacent motifs (PAM sequence). Combined with sequence motif labeling, we can pinpoint the single-base differences in highly conserved sequences. In the second strategy, we design mapping patterns across a genome by selecting sets of specific single-guide RNAs (sgRNAs) for labeling multiple loci of a genomic region or a whole genome. By developing and optimizing a single tube synthesis of multiple sgRNAs, we demonstrate the utility of CRISPR-Cas9 mapping with 162 sgRNAs targeting the 2Mb Haemophilus influenzae chromosome. These CRISPR-Cas9 mapping approaches could be particularly useful for applications in defining long-distance haplotypes and pinpointing the breakpoints in large structural variants in complex genomes and microbial mixtures.

High-throughput single-molecule imaging system using nanofabricated trenches and fluorescent DNA-binding proteins.

DNA curtain is a high-throughput system, integrating a lipid bilayer, fluorescence imaging, and microfluidics to probe protein-DNA interactions in real-time and has provided in-depth understanding of DNA metabolism. Especially, the microfluidic platform of a DNA curtain is highly suitable for a biochip. In the DNA curtain, DNA molecules are aligned along chromium nanobarriers, which are fabricated on a slide surface, and visualized using an intercalating dye, YOYO-1. Although the chromium barriers confer precise geometric alignment of DNA, reuse of the slides is limited by wear of the barriers during cleaning. YOYO-1 is rapidly photobleached and causes photocleavage of DNA under continuous laser illumination, restricting DNA observation to a brief time window. To address these challenges, we developed a new nanopatterned slide, upon which carved nanotrenches serve as diffusion barriers. The nanotrenches were robust under harsh cleaning conditions, facilitating the maintenance of surface cleanliness that is essential to slide reuse. We also stained DNA with a fluorescent protein with a DNA-binding motif, fluorescent protein-DNA binding peptide (FP-DBP). FP-DBP was slowly photobleached and did not cause DNA photocleavage. This new DNA curtain system enables a more stable and repeatable investigation of real-time protein-DNA interactions and will serve as a good platform for lab-on-a-chip.

Oxidative Effects during Irreversible Electroporation of Melanoma Cells-In Vitro Study.

Irreversible electroporation (IRE) is today used as an alternative to surgery for the excision of cancer lesions. This study aimed to investigate the oxidative and cytotoxic effects the cells undergo during irreversible electroporation using IRE protocols. To do so, we used IRE-inducing pulsed electric fields (PEFs) (eight pulses of 0.1 ms duration and 2-4 kV/cm intensity) and compared their effects to those of PEFs of intensities below the electroporation threshold (eight pulses, 0.1 ms, 0.2-0.4 kV/cm) and the PEFs involving elongated pulses (eight pulses, 10 ms, 0.2-0.4 kV/cm). Next, to follow the morphology of the melanoma cell membranes after treatment with the PEFs, we analyzed the permeability and integrity of their membranes and analyzed the radical oxygen species (ROS) bursts and the membrane lipids' oxidation. Our data showed that IRE-induced high cytotoxic effect is associated both with irreversible cell membrane disruption and ROS-associated oxidation, which is occurrent also in the low electric field range. It was shown that the viability of melanoma cells characterized by similar ROS content and lipid membrane oxidation after PEF treatment depends on the integrity of the membrane system. Namely, when the effects of the PEF on the membrane are reversible, aside from the high level of ROS and membrane oxidation, the cell does not undergo cell death.

Optimization of the droplet electroporation method for wild type Chlamydomonas reinhardtii transformation.

We performed the transformation of a wild type Chlamydomonas reinhardtii by optimizing previously developed droplet EP method. For more effective and faster optimization, we used DNA dying fluorescent molecule (Yo-Pro-1) for finding optimal EP conditions instead of using protein expression based evaluation method. By examining wider range of electrical parameter space together with the analysis of total current flow of EP process, we found optimal EP conditions. The obtained optimal EP conditions were verified by CFP transgene expression experiments. By applying the optimal EP conditions to the transformation of C. reinhardtii, we obtained transformants and analyzed them using PCR. Finally, implications and future work are discussed.

High fidelity visualization of cell-to-cell variation and temporal dynamics in nascent extracellular matrix formation.

Extracellular matrix dynamics are key to tissue morphogenesis, homeostasis, injury, and repair. The spatiotemporal organization of this matrix has profound biological implications, but is challenging to monitor using standard techniques. Here, we address these challenges by using noncanonical amino acid tagging to fluorescently label extracellular matrix synthesized in the presence of bio-orthogonal methionine analogs. This strategy labels matrix proteins with high resolution, without compromising their distribution or mechanical function. We demonstrate that the organization and temporal dynamics of the proteinaceous matrix depend on the biophysical features of the microenvironment, including the biomaterial scaffold and the niche constructed by cells themselves. Pulse labeling experiments reveal that, in immature constructs, nascent matrix is highly fibrous and interdigitates with pre-existing matrix, while in more developed constructs, nascent matrix lacks fibrous organization and is retained in the immediate pericellular space. Inhibition of collagen crosslinking increases matrix synthesis, but compromises matrix organization. Finally, these data demonstrate marked cell-to-cell heterogeneity amongst both chondrocytes and mesenchymal stem cells undergoing chondrogenesis. Collectively, these results introduce fluorescent noncanonical amino acid tagging as a strategy to investigate spatiotemporal matrix organization, and demonstrate its ability to identify differences in phenotype, microenvironment, and matrix assembly at the single cell level.

Studies on visual detection and surface modification testing of glass microfiber filter paper based biosensor.

Glass microfibers are commonly used as biomolecule adsorption media, as structural or disposable components of the optical biosensors. While any improvement in these components are appreciated, utilizing basic tools of traditional approaches may lead to original sensor opportunities as simple, functional designs that can be easily disseminated. Following this pursuit, surface modification of glass microfiber paper surface was performed by 3-aminopropyltriethoxysilane (APTES) and resulting improvement in the cell entrapment capacity could be observed visually, only after Gram staining. Gram staining offered rapid validation of enhanced binding on the glass surface. The same APTES-modified samples were also tested for binding of complementary DNA sequences and the results were less straightforward due to the necessity of DNA visualization by using a fluorescent stain, YOYO-1. Accordingly, when there were no surface modification, DNA and YOYO-1 adsorbed readily on the glass microfiber filter paper, and prolonged the interaction between DNA and YOYO-1. YOYO-1 adsorption on glass could be recognized from the color profile of YOYO-1 emission. This phenomenon can be used to examine suitability of APTES coverage on glass surfaces since YOYO-1 emission can be distinguished by its glass adsorbed versus DNA-bound forms. Aptness of surface coverage is vital to biosensor studies in the sense that it is preceding the forthcoming surface modifications and its precision is imperative for attaining the anticipated interaction kinetics of the surface-immobilized species. The proposed testing scheme offered in this study secures the work, which is aimed to be carried out utilizing such sensing systems and device components.

Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad51.

Low throughput is an inherent problem associated with most single-molecule biophysical techniques. We have developed a versatile tool for high-throughput analysis of DNA and DNA-binding molecules by combining microfluidic and dense DNA arrays. We use an easy-to-process microfluidic flow channel system in which dense DNA arrays are prepared for simultaneous imaging of large amounts of DNA molecules with single-molecule resolution. The Y-shaped microfluidic design, where the two inlet channels can be controlled separately and precisely, enables the creation of a concentration gradient across the microfluidic channel as well as rapid and repeated addition and removal of substances from the measurement region. A DNA array stained with the fluorescent DNA-binding dye YOYO-1 in a gradient manner illustrates the method and serves as a proof of concept. We have applied the method to studies of the repair protein Rad51 and could directly probe the concentration-dependent DNA-binding behavior of human Rad51 (HsRad51). In the low-concentration regime used (100 nM HsRad51 and below), we detected binding to double-stranded DNA (dsDNA) without positive cooperativity.

Electroporation of adherent cells with low sample volumes on a microscope stage.

The labeling of specific molecules and their artificial control in living cells are powerful techniques for investigating intracellular molecular dynamics. To use these techniques, molecular compounds (hereinafter described simply as 'samples') need to be loaded into cells. Electroporation techniques are exploited to load membrane-impermeant samples into cells. Here, we developed a new electroporator with four special characteristics. (1) Electric pulses are applied to the adherent cells directly, without removing them from the substratum. (2) Samples can be loaded into the adherent cells while observing them on the stage of an inverted microscope. (3) Only 2 µl of sample solution is sufficient. (4) The device is very easy to use, as the cuvette, which is connected to the tip of a commercially available auto-pipette, is manipulated by hand. Using our device, we loaded a fluorescent probe of actin filaments, Alexa Fluor 546 phalloidin, into migrating keratocytes. The level of this probe in the cells could be easily adjusted by changing its concentration in the electroporation medium. Samples could be loaded into keratocytes, neutrophil-like HL-60 cells and Dictyostelium cells on a coverslip, and keratocytes on an elastic silicone substratum. The new device should be useful for a wide range of adherent cells and allow electroporation for cells on various types of the substrata.

Flow cytometry as a tool for analyzing changes in Plasmodium falciparum cell cycle following treatment with indol compounds.

Melatonin and its derivatives modulate the Plasmodium falciparum and Plasmodium chabaudi cell cycle. Flow cytometry was employed together with the nucleic acid dye YOYO-1 allowing precise discrimination between mono- and multinucleated forms of P. falciparum-infected red blood cell. The use of YOYO-1 permitted excellent discrimination between uninfected and infected red blood cells as well as between early and late parasite stages. Fluorescence intensities of schizont-stage parasites were about 10-fold greater than those of ring-trophozoite form parasites. Melatonin and related indolic compounds including serotonin, N-acetyl-serotonin and tryptamine induced an increase in the percentage of multinucleated forms compared to non-treated control cultures. YOYO-1 staining of infected erythrocyte and subsequent flow cytometry analysis provides a powerful tool in malaria research for screening of bioactive compounds.

Benzothiazole-substituted benzofuroquinolinium dye: a selective switch-on fluorescent probe for G-quadruplex.

A new switch-on fluorescent probe containing the natural product cryptolepine analogue benzofuroquinolinium moiety (binding scaffold) and a benzothiazole moiety (signalling unit) shows a remarkable fluorescence enhancement selective for the G-quadruplex nucleic acid structure. Binding studies revealed that the highly selective response of the fluorescent probe arises from end-stack binding to G-quadruplex.

Screening by imaging: scaling up single-DNA-molecule analysis with a novel parabolic VA-TIRF reflector and noise-reduction techniques.

Bacteriophage lambda-DNA molecules are frequently used as a scaffold to characterize the action of single proteins unwinding, translocating, digesting or repairing DNA. However, scaling up such single-DNA-molecule experiments under identical conditions to attain statistically relevant sample sizes remains challenging. Additionally the movies obtained are frequently noisy and difficult to analyse with any precision. We address these two problems here using, firstly, a novel variable-angle total internal reflection fluorescence (VA-TIRF) reflector composed of a minimal set of optical reflective elements, and secondly, using single value decomposition (SVD) to improve the signal-to-noise ratio prior to analysing time-lapse image stacks. As an example, we visualize under identical optical conditions hundreds of surface-tethered single lambda-DNA molecules, stained with the intercalating dye YOYO-1 iodide, and stretched out in a microcapillary flow. Another novelty of our approach is that we arrange on a mechanically driven stage several capillaries containing saline, calibration buffer and lambda-DNA, respectively, thus extending the approach to high-content, high-throughput screening of single molecules. Our length measurements of individual DNA molecules from noise-reduced kymograph images using SVD display a 6-fold enhanced precision compared to raw-data analysis, reaching approximately 1 kbp resolution. Combining these two methods, our approach provides a straightforward yet powerful way of collecting statistically relevant amounts of data in a semi-automated manner. We believe that our conceptually simple technique should be of interest for a broader range of single-molecule studies, well beyond the specific example of lambda-DNA shown here.

Development and validation of flow cytometric measurement for parasitaemia using autofluorescence and YOYO-1 in rodent malaria.

An automated flow cytometric (FCM) detection method has been developed and validated with a simple diagnostic procedure in parasitized erythrocytes of Plasmodium berghei-infected rats using the nucleic acid-binding fluorescent dye YOYO-1. High levels of reticulocytes were detected during the course of the infection, ranging from 1.2-51.2%, but any RNA potentially confounding the assay could be removed by digestion with RNAse. The cell counts of uninfected, infected, and nucleated cells occurred with high precision. The cells were divided into different populations according to their physical or chemical properties but various factors within the assay such as cell fixation, RNA digestion, and compensation required optimization. In this study, FCM greatly simplified and accelerated parasite detection, with a mean precision of 4.4%, specificity of 98.9% and accuracy of 101.3%. The detection and quantitation limits in the assay were 0.024% and 0.074% parasitaemia, respectively. Overall, the parasite counts by FCM measurement correlated highly (r2=0.954-0.988) with the parasitaemia measured by light microscopical analysis when animals treated with suppressive, clearance, and curative doses of novel antimalarial drugs were examined. The lower levels of parasitaemia (30%) detected by microscopy compared to FCM may be related to a number of schizonts externally attached to the erythrocyte membranes that normally would not be included in microscopy counting. Lower sampling error and reliable identification of rodent erythrocyte parasites based on the principles of FCM have replaced the traditional blood smear in our laboratory.

Development and validation of flow cytometric measurement for parasitemia in cultures of P. falciparum vitally stained with YOYO-1.

BACKGROUND: The need for improved malaria diagnostics has long been recognized. METHODS: Human parasitized erythrocytes based on the principles of flow cytometry (FCM) method is described for the determination of parasitemia in Plasmodium falciparum cultures using the fluorescence activated cell sorter and DNA-binding fluorescent dye, YOYO-1. The same assay samples can be also evaluated both microscopically and by scintillation counting after use of (3)H-hypoxanthine-labeled parasites. RESULTS: The counts of uninfected, infected, and nucleated cells occurred with high precision. The cells were categorized into different populations according to their physical or chemical properties such as RNase treatment and compensation required optimization. The detection and quantitation limits in the assay were 0.003% and 0.008% parasitemia, respectively. Overall, the parasite counts by FCM measurement correlated highly (r(2) = 0.923-0.968) with the parasitemia measured by (3)H-hypoxanthine incorporation assay when parasites variants incubated with various antimalarial drugs. In addition, the low levels of parasitemia (7.9%-21.3%) detected by microscopy than by FCM may be related to a number of tiny schizonts externally attached to the erythrocyte membranes but were not definitely inside the erythrocyte that normally would never be included in microscopy counting. CONCLUSION: On the basis of data reported herein, a rapid, high sensitivity, lower sampling error and reliable identification of human parasitized erythrocytes by the principles of FCM have been established. Published 2007 Wiley-Liss, Inc.

Increased intensities of YOYO-1-labeled DNA oligomers near silver particles.

DNA detection is usually performed using fluorescence probes. Using a DNA oligomer stained with the widely used dye 1,1'-[1,3-propanediylbis[(dimethylimino)-3,1-propanediyl]]bis[4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]]-quinolinum tetraiodide (YOYO-1), we show that a substrate containing silver particles can lead to a greater than 10-fold increase in the fluorescence intensity. Proximity to silver particles also increases the photostability of YOYO-1-DNA. These results suggest that substrates or gels containing silver particles may be used for increased sensitivity in DNA detection.

Catalytic enantioselective Reissert-type reaction: development and application to the synthesis of a potent NMDA receptor antagonist (-)-L-689,560 using a solid-supported catalyst.

Full details of the first catalytic enantioselective Reissert-type reaction are described. Utilizing the Lewis acid-Lewis base bifunctional catalyst 5 or 6 (9 mol %), the Reissert products were obtained in 57 to 99% yield with 54 to 96% ee. Electron-rich quinolines produced better yields and enantioselectivities than electron-deficient substrates. Kinetic studies indicated that the reaction should proceed via the rate-determining acyl quinolinium formation, followed by the attack of a cyanide. The catalyst does not facilitate the first rate-determining step; however, it strongly facilitates the second cyanation step. The reaction was successfully applied to an efficient catalytic asymmetric synthesis of a potent NMDA receptor antagonist (-)-L-689,560. A key step is the one-pot process using the Reissert-type reaction from quinoline 1f, followed by stereoselective reduction of the resulting enamine 2f. This step gave the key intermediate 20 in 91% yield with 93% ee, using 1 mol % of 6. The enantiomerically pure target compound was obtained through 10 operations (including recrystallization) in total yield of 47%. Furthermore, 6 was immobilized to JandaJEL, and the resulting solid-supported catalyst 11 afforded 20 in a comparable yield to the homogeneous 6, but with slightly lower enantioselectivity.

Chloride-sensitive fluorescent indicators.

Three fluorescent halide-sensitive quinolinium dyes have been produced by the reaction of the 6-methylquinoline heterocyclic nitrogen base with methyl bromide, methyl iodide, and 3-bromo-1-propanol. The quaternary salts, unlike the precursor molecule, are readily water soluble and the fluorescence intensity of these salts is reduced in the presence of aqueous chloride, bromide, and iodide ions, allowing halide solution concentrations to be determined using well-known Stern-Volmer kinetics. One of the dyes, dye 1, has a chloride Stern-Volmer constant of 255 mol(-1) dm(3) which is more than twice that of SPQ [6-methoxy-N-(3-sulfopropyl)quinolinium] used in recent physiological measurements to measure intracellular chloride levels. The dyes have been characterized using steady-state fluorescence spectroscopy and are compared to three similar dyes based on the 6-methoxyquinoline nucleus, reported earlier by the authors, and also to dyes reported by Krapf et al. (Anal. Biochem. 169, 142-150, 1988). The interference of aqueous anions and the potential for using these dyes in biological halide-sensing applications are discussed.

Electrophoresis in microfabricated devices using photopolymerized polyacrylamide gels and electrode-defined sample injection.

Microfabrication techniques have become increasingly popular in the development of the next generation of DNA analysis systems. While significant progress has been reported by many researchers, complete microfabricated integrated DNA analysis devices are still in the earliest stages of development. Most miniaturized analysis systems have incorporated noncross-linked polymer solutions as the separation medium of choice and the operation of these systems necessitates the use of high electric fields and long separation lengths. In this paper, we present two techniques that may help alleviate this problem and accelerate the development of the so-called 'lab-on-a-chip' systems. We present the use of photodefinable polyacrylamide gels as a sieving medium for DNA electrophoresis. These gels offer the significant advantages of faster curing times, locally controlled gel interface, and simpler handling over chemically polymerized gels. We also introduce an electrode-defined sample compaction and injection technique. This technique helps achieve sample compaction without migration into the gel and offers significant control over the size and application of the sample plug. The use of these technologies for double-stranded DNA separations in microfabricated separation systems is demonstrated.

Measurement of chloride movement in neuronal preparations.

In this unit, protocols are described for biochemical and optical techniques that have been used by investigators to measure ligand-gated chloride movement in vesicular structures called synaptoneurosomes (also referred to as microsacs), in cultured neurons, and in the acute brain slice. These techniques can be applied to other ions as well. The measurement of uptake and efflux of radioisotopic chloride in synaptoneurosomes is used to study the responses of gamma-aminobutyric acid (GABA) receptors, which are coupled to chloride channels. Similar chloride flux assays for primary neuronal cultures are also presented. Alternatively, the efflux of chloride from synaptoneurosomes and primary neuronal cultures can be studied using fluorescent dyes and photometry. Finally, the measurement of chloride uptake can be studied in individual neurons in brain slices using fluorescent dyes and optical imaging by nonconfocal and confocal microscopy. Several support protocols are provided as well, outlining the preparation of synaptoneurosomes from specific brain regions, and the preparation, loading, and calibration of chloride-sensitive fluorescent dyes.

Optimization of intercalation dye concentration for short tandem repeat allele genotyping using capillary electrophoresis with laser-induced fluorescence detection.

DNA analysis using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection requires that polymerase chain reaction products either be prepared using primers with fluorescent molecules covalently bonded to them, or stained with a fluorescent intercalation dye following amplification. The intercalation technique has the advantage of allowing fluorescence detection of any double-stranded DNA (dsDNA) product regardless of the amplification primers used. The increased sensitivity of LIF detection is sometimes compromised by the intercalation dye changing the mass to charge ratio of the DNA. The purpose of this study was to evaluate the changes of migration rate, resolution and fluorescent intensity of dye-DNA complexes during electrophoretic separations, and to establish the optimal parameters for short tandem repeats alleles profiling. The alleles of three STR loci THO1, F13A01 and vWFA31 were intercalated with the monomeric dyes TOPRO-1 and YOPRO-1, and their corresponding dimers, TOTO-1 and YOYO-1 (Molecular Probes, Eugene, OR, USA). Alleles intercalated before injection onto the CE column resulted in loss of resolution and sensitivity when compared to the on-column labeling technique. The results of this experimentation were then applied to a STR typing assay using a commercially available polymer and buffer matrix. This assay included development of a unique internal standard used for migration time normalization assignment of alleles. Consequently, the 9 allele and the 9.3 microvariant of the THOI locus were separated and typed correctly with a resolution of 0.49 in less than 20 min, and the only sample preparation necessary after amplification was a dilution step.

Isocyanines and pseudoisocyanines as a novel class of potent noradrenaline transport inhibitors: synthesis, detection, and biological activity.

Noradrenaline, the neurotransmitter of the sympathetic nervous system, is removed from the extracellular space by both neuronal and extraneuronal transport mechanisms. In the past, further investigation of the extraneuronal type of noradrenaline transporter was severely hampered by the lack of potent inhibitors. Here, we describe the synthesis of various novel noradrenaline transport inhibitors which belong to the chemical class of isocyanine and pseudoisocyanine dyes. The biological activity of these compounds was investigated in a tissue culture system (Caki-1 cells). 1,1'-Diisopropyl-2,4'-cyanine, 1,1'-diethyl-2,2'-cyanine, and 1-ethyl-1'-isopropyl-2,2'-cyanine turned out as the most potent inhibitors of the extraneuronal noradrenaline transport known so far. At 100 nmol/L, these compounds diminished extraneuronal noradrenaline transport by about 95%. Their IC50's were below 20 nmol/L. In addition, a rapid and sensitive method (based on HPLC with fluorometric detection) to measure these compounds in body fluids is reported.