Isothermal Assay Research Articles

Isoform-Specific Inhibition of Cyclophilins

Cyclophilins belong to the enzyme class of peptidyl prolyl cis-trans isomerases which catalyze the cis-trans isomerization of prolyl bonds in peptides and proteins in different folding states. Cyclophilins have been shown to be involved in a multitude of cellular functions like cell growth, proliferation, and motility. Among the 20 human cyclophilin isoenzymes, the two most abundant members of the cyclophilin family, CypA and CypB, exhibit specific cellular functions in several inflammatory diseases, cancer development, and HCV replication. A small-molecule inhibitor on the basis of aryl 1-indanylketones has now been shown to discriminate between CypA and CypB in vitro. CypA binding of this inhibitor has been characterized by fluorescence anisotropy- and isothermal titration calorimetry-based cyclosporin competition assays. Inhibition of CypA- but not CypB-mediated chemotaxis of mouse CD4(+) T cells by the inhibitor provided biological proof of discrimination in vivo.

Development of Isothermal TaqMan Assays for Detection of Biothreat Organisms

TaqMan probe (dual-labeled DNA probe)-based real-time detection, one of the most sensitive and specific fluorescent detection methods, has been widely utilized in conjunction with polymerase chain reaction (PCR). Helicase-dependent amplification (HDA) is an isothermal amplification technology that has a similar reaction scheme to PCR, but replaces thermocycling with a helicase capable of unwinding a DNA duplex. Here we describe a novel isothermal real-time detection method (HDA-TaqMan) that combines the advantages of both HDA and a TaqMan assay. In this assay, the reactions of DNA unwinding, primer annealing, polymerization, probe hybridization, and subsequent hydrolysis by the polymerase are coordinated and synchronized to perform at a single temperature. It not only provides a useful tool for real-time detection of HDA, but also provides an isothermal format for the TaqMan system. With this platform, we have successfully developed rapid real-time isothermal assays for biodefense targets that include Vibrio cholerae and Bacillus anthracis.

Calcium Binds to Leptospiral Immunoglobulin-like Protein, LigB, and Modulates Fibronectin Binding

Pathogenic Leptospira spp. express immunoglobulin-like proteins, LigA and LigB, which serve as adhesins to bind to extracellular matrices and mediate their attachment on host cells. However, nothing is known about the mechanism by which these proteins are involved in pathogenesis. We demonstrate that LigBCen2 binds Ca(2+), as evidenced by inductively coupled plasma optical emission spectrometry, energy dispersive spectrometry, (45)Ca overlay, and mass spectrometry, although there is no known motif for Ca(2+) binding. LigBCen2 binds four Ca(2+) as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The dissociation constant, K(D), for Ca(2+) binding is 7 mum, as measured by isothermal titration calorimetry and calcium competition experiments. The nature of the Ca(2+)-binding site in LigB is possibly similar to that seen in the betagamma-crystallin superfamily, since structurally, both families of proteins possess the Greek key type fold. The conformation of LigBCen2 was significantly influenced by Ca(2+) binding as shown by far- and near-UV CD and by fluorescence spectroscopy. In the apo form, the protein appears to be partially unfolded, as seen in the far-UV CD spectrum, and upon Ca(2+) binding, the protein acquires significant beta-sheet conformation. Ca(2+) binding stabilizes the protein as monitored by thermal unfolding by CD (50.7-54.8 degrees C) and by differential scanning calorimetry (50.0-55.7 degrees C). Ca(2+) significantly assists the binding of LigBCen2 to the N-terminal domain of fibronectin and perturbs the secondary structure, suggesting the involvement of Ca(2+) in adhesion. We demonstrate that LigB is a novel bacterial Ca(2+)-binding protein and suggest that Ca(2+) binding plays a pivotal role in the pathogenesis of leptospirosis.

Simple, rapid and sensitive detection of Orientia tsutsugamushi by loop-isothermal DNA amplification

We present a loop-mediated isothermal PCR assay (LAMP) targeting the groEL gene, which encodes the 60kDa heat shock protein of Orientia tsutsugamushi. Evaluation included testing of 63 samples of contemporary in vitro isolates, buffy coats and whole blood samples from patients with fever. Detection limits for LAMP were assessed by serial dilutions and quantitation by real-time PCR assay based on the same target gene: three copies/microl for linearized plasmids, 26 copies/microl for VERO cell culture isolates, 14 copies/microl for full blood samples and 41 copies/microl for clinical buffy coats. Based on a limited sample number, the LAMP assay is comparable in sensitivity with conventional nested PCR (56kDa gene), with limits of detection well below the range of known admission bacterial loads of patients with scrub typhus. This inexpensive method requires no sophisticated equipment or sample preparation, and may prove useful as a diagnostic assay in financially poor settings; however, it requires further prospective validation in the field setting.

Rapid isothermal detection assay: a probe amplification method for the detection of nucleic acids

Simple, accurate, and stable diagnostic tests are essential to control viral infectious diseases such as avian influenza virus. The current technologies are often inaccessible to people who need them, mainly because of the specialized equipment and the need for highly trained technologists. Here, we describe a rapid isothermal nucleic acid detection assay (RIDA) that can be used to detect both DNA and RNA targets. Using chemically modified probes, we designed a lateral-flow (LF) immunoassay that can be used in combination with RIDA for equipment-free nucleic acid target detection. RIDA is a “probe amplification” assay that uses the single-strand nicking activity of restriction nicking endonucleases to repeatedly cleave synthetic probes hybridizing to the same target sequences. In the RIDA-LF combined assay, chemically labeled probes are covalently conjugated to magnetic microbeads, which is propitious to separate cleaved probes from the reaction solution. The cleaved probes in the solution are then detected with an LF immunoassay. The real-time assay shows that RIDA is able to specifically detect target sequences in 5 to 15 min. The RIDA-LF combined assay can specifically detect nucleic acid targets without sophisticated equipment. In this report, our data suggest that RIDA is a flexible simple assay that could be applied for point-of-care detection. The modified-RIDA described in this report further extends the application of this technology.

Effects of trehalose click polymer length on pDNA complex stability and delivery efficacy

Cationic polymers are currently being studied as non-viral vectors to deliver therapeutic DNA into cells. In this study, a series of trehalose-based glycopolymers containing four secondary amines in the repeat unit were synthesized via the ‘click reaction’ [degrees of polymerization ( n w)=35, 53, 75, or 100] to elucidate how the polymer length affects the bioactivity. The four structures bound and charge-neutralized pDNA with similar affinity that was independent of the length, as determined through gel electrophoresis, heparin competitive displacement, and isothermal titration calorimetric assays. Dynamic light scattering measurements revealed that the polyplexes formed with the longer polymers ( n w=53, 75, or 100) inhibited flocculation in media containing serum, whereas the polyplexes formed with the shorter polymer ( n w=35) aggregated rapidly. Similar results were observed via transmission electron microscopy studies, where the nanoparticles formed with the polymers having longer degrees of polymerization showed discrete particles in media containing 10% serum. Transfection experiments revealed that the polymers exhibited low cytotoxicity at low N/P ratios and could facilitate high cellular uptake and gene expression in HeLa and H9c2(2-1) cells, and the results were dependent on the degrees of polymerization (longer polymers yielded higher transfection and toxicity).

Genotype-Specific Signal Generation Based on Digestion of 3-Way DNA Junctions: Application to KRAS Variation Detection

Genotyping methods that reveal single-nucleotide differences are useful for a wide range of applications. We used digestion of 3-way DNA junctions in a novel technology, OneCutEventAmplificatioN (OCEAN) that allows sequence-specific signal generation and amplification. We combined OCEAN with peptide-nucleic-acid (PNA)-based variant enrichment to detect and simultaneously genotype v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) codon 12 sequence variants in human tissue specimens. We analyzed KRAS codon 12 sequence variants in 106 lung cancer surgical specimens. We conducted a PNA-PCR reaction that suppresses wild-type KRAS amplification and genotyped the product with a set of OCEAN reactions carried out in fluorescence microplate format. The isothermal OCEAN assay enabled a 3-way DNA junction to form between the specific target nucleic acid, a fluorescently labeled "amplifier", and an "anchor". The amplifier-anchor contact contains the recognition site for a restriction enzyme. Digestion produces a cleaved amplifier and generation of a fluorescent signal. The cleaved amplifier dissociates from the 3-way DNA junction, allowing a new amplifier to bind and propagate the reaction. The system detected and genotyped KRAS sequence variants down to approximately 0.3% variant-to-wild-type alleles. PNA-PCR/OCEAN had a concordance rate with PNA-PCR/sequencing of 93% to 98%, depending on the exact implementation. Concordance rate with restriction endonuclease-mediated selective-PCR/sequencing was 89%. OCEAN is a practical and low-cost novel technology for sequence-specific signal generation. Reliable analysis of KRAS sequence alterations in human specimens circumvents the requirement for sequencing. Application is expected in genotyping KRAS codon 12 sequence variants in surgical specimens or in bodily fluids, as well as single-base variations and sequence alterations in other genes.

Bleaching of kaolins and clays by chlorination of iron and titanium

The quality of the clays and over all kaolin is measured in function of iron content, since this element gives an undesirable reddish color to this type of minerals. The use of chlorination for iron and titanium removal from different clay and kaolin minerals, used in Argentinian ceramic industry, has been investigated to establish reaction mechanisms, precautions and optimal conditions to bleach the clays. The method consists of the calcinations of the pellets in a flow of chlorine gas at temperatures between 700 and 950 °C, to remove the iron and the titanium by volatilization of the respective chlorides. Isothermal and non-isothermal chlorination assays were made and the effects of the temperature, reaction time, and carbon content in the sample over the bleach of the minerals and the phase transformations suffered by these minerals during the chlorination step were investigated. The removed amounts of iron and titanium were determined by X-ray fluorescence, the phase transformations were followed by X-ray diffraction and the samples' bleaching was established making an analysis of the space of the color by the spectrophofotometric method CIELAB. Also, a thermodynamic analysis of the system using HSC Chemistry for Windows software was made. The experimental results show that for red clay with high colloidal iron content, the optimum working temperature for the iron quantitative removal, without aluminium loss, is close to 850 °C, in controlled atmosphere free of O 2 and H 2O; lower temperatures require long reaction times. The extraction of titanium is not quantitatively achieved, as that of iron, even for long reaction times, because this element is present in different structures, with the consequent variation of its reactivity. The content of carbon and organic matter in the clays, in the case of the San Luis red clay, is enough for extraction improvement. However, the optimum concentration of carbon can vary depending on the type of clay and the amount of Fe 2O 3 in the mineral. The formation of phases such as α-Al 2O 3, a product of chlorination, increases the piece hardness. The methodology used in this work allows for obtaining a great improvement in the color of the final product of the firing, achieving ceramic materials with notable whiteness, even with red clays.

Specificity in DNA recognition by a peptide from papillomavirus E2 protein

The E2 proteins of papillomavirus specifically bind to double-stranded DNA containing the consensus sequence ACCG-N 4-CGGT, where N is any nucleotide. Here, we show the binding and recognition of dissimilar DNA sequences by an 18 amino-acid peptide (α1E2), which corresponds to the DNA-recognition helix, α-helix-1. Isothermal DNA binding assays performed with the DNA consensus sequence show saturable curves with α1E2 peptide, and the α1E2 peptide is converted to an ordered conformation upon complexation. Measurements performed with non-specific DNA sequence fail to saturate, a behavior characteristic of non-specific binding. Binding of the α1E2 peptide to these DNA sequences display a different counter-ion dependence, indicating a dissimilar, sequence-dependent mechanism of interaction. Quantitative stoichiometric measurements revealed the specificity in α1E2 peptide recognition of the ACCG half-site, demonstrating capacity for discrimination of nucleic acid bases sequences without the need of a whole protein architecture.

Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction.

The invasive signal amplification reaction has been previously developed for quantitative detection of nucleic acids and discrimination of single-nucleotide polymorphisms. Here we describe a method that couples two invasive reactions into a serial isothermal homogeneous assay using fluorescence resonance energy transfer detection. The serial version of the assay generates more than 10(7) reporter molecules for each molecule of target DNA in a 4-h reaction; this sensitivity, coupled with the exquisite specificity of the reaction, is sufficient for direct detection of less than 1,000 target molecules with no prior target amplification. Here we present a kinetic analysis of the parameters affecting signal and background generation in the serial invasive signal amplification reaction and describe a simple kinetic model of the assay. We demonstrate the ability of the assay to detect as few as 600 copies of the methylene tetrahydrofolate reductase gene in samples of human genomic DNA. We also demonstrate the ability of the assay to discriminate single base differences in this gene by using 20 ng of human genomic DNA.

A steep thermal gradient thermotaxis assay for the nematode Caenorhabditis elegans.

The nematode Caenorhabditis elegans with its well-described nervous system is one of the multicellular organisms of choice to study thermotaxis. The neuronal circuitry for thermosensation has been analyzed at the level of individual cells. Two methods have previously been described to study the behavior of C. elegans with respect to temperature: 1) isothermal tracking assays and 2) linear thermal gradients (Hedgecock and Russell, 1975). Here we present a short linear thermal gradient assay which is faster and which allows statistical evaluation of different populations using a thermotaxis index. Thin agar plates are used on which a temperature gradient from about 10 degrees to 30 degrees is induced over the distance of about 5 cm. The short linear thermal gradient uses inexpensive materials so that multiple tests can be performed in parallel in a short period of time.

Lack of usefulness of DN-1417 for characterization of a CNS receptor for thyrotropin-releasing hormone.

CNS receptors for thyrotropin-releasing hormone (TRH) and its analogs are likely to mediate the experimentally and clinically observed net excitatory effect of these peptides on lower motor neurons. Previous findings suggest that several types of TRH receptors with distinct TRH analog specificities may be present in rat CNS. In particular, based on competition isotherm assays with unlabeled analog gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolineamide (DN-1417). Funatsu et al. claim the existence of a limbic forebrain site that binds this peptide and TRH with high affinity but that does not bind [3-methyl-histidyl2]-TRH (MeTRH). Using saturation and competition isotherm experiments, we have examined the binding of [3H]TRH and [3H]DN-1417 in three regions of rat CNS: pyriform cortex/amygdala, limbic forebrain, and lumbosacral spinal cord. In all three regions, saturation assays with [3H]TRH (0.4-100 nM) resolved only a single, saturable receptor with high affinity (KD = 12-14 nM) for TRH; in no case could more than one saturable site be identified. When [3H]DN-1417 was substituted as the assay ligand, no high-affinity binding component for this analog could be detected in the three regions. Competition curves for the binding of unlabeled DN-1417 to limbic forebrain and lumbosacral spinal cord ([3H]TRH as assay ligand) were monophasic (not biphasic like those of Funatsu et al.) and indicative of low-affinity binding of DN-1417 in these regions (Ki values = 2-3 microM; in agreement with values obtained in similar assays with [3H]MeTRH).(ABSTRACT TRUNCATED AT 250 WORDS)