Target sequence heterogeneity causes the 'hook effect' in fluorescent dye-based quantitative PCR.

The 'hook effect' describes a phenomenon in quantitative PCR (qPCR) amplification curves where fluorescence values decrease following an initial amplification phase. We propose that in intercalating dye-based qPCR, the 'hook effect' is due to the amplification of heterogeneous but related DNA targets. The decrease in fluorescence at later cycles occurs because the related products self-anneal to form a DNA heteroduplex with a melt temperature below the temperature at which the fluorescence measurement is made. We show this experimentally using qPCR of Alu family repetitive DNA elements.

The Lysosomotropic Activity of Hydrophobic Weak Base Drugs is Mediated via Their Intercalation into the Lysosomal Membrane.

Lipophilic weak base therapeutic agents, termed lysosomotropic drugs (LDs), undergo marked sequestration and concentration within lysosomes, hence altering lysosomal functions. This lysosomal drug entrapment has been described as luminal drug compartmentalization. Consistent with our recent finding that LDs inflict a pH-dependent membrane fluidization, we herein demonstrate that LDs undergo intercalation and concentration within lysosomal membranes. The latter was revealed experimentally and computationally by (a) confocal microscopy of fluorescent compounds and drugs within lysosomal membranes, and (b) molecular dynamics modeling of the pH-dependent membrane insertion and accumulation of an assortment of LDs, including anticancer drugs. Based on the multiple functions of the lysosome as a central nutrient sensory hub and a degradation center, we discuss the molecular mechanisms underlying the alteration of morphology and impairment of lysosomal functions as consequences of LDs' intercalation into lysosomes. Our findings bear important implications for drug design, drug induced lysosomal damage, diseases and pertaining therapeutics.

Integrated Optoelectronic Device for Detection of Fluorescent Molecules.

This paper presents the development of a compact optoelectronic device suitable for on-chip detection of fluorescent molecules. In order to obtain a highly integrated device, a long-pass multi-dielectric filter has been integrated with thin-film amorphous silicon photosensors on a single glass substrate. Filter rejects the excitation light, allowing the reduction of the distance between the source and the fluorescent site and avoiding the use of external optical component. The compatibility of the technological processes determined the materials and the temporal sequence of the device fabrication. The developed device has been designed for the fluorescence detection of ruthenium complex based molecules and tested, as a proof of concept, for the detection of double-stranded DNA down to 0.5 ng. Results demonstrate the correct operation of the integrated system in both rejecting the excitation light and in detecting the fluorescent signal, demonstrating the suitability of this optoelectronic platform in practical biomedical applications.

T-blocker: a simple and robust probe-free quantitative PCR assay to detect somatic mutations down to 0.1% frequency.

We have developed a simple and robust probe-free quantitative PCR (qPCR) assay method that can detect minor mutant alleles with a frequency as low as 0.1% in a heterogeneous sample by introducing a novel T-blocker concept to the allele-specific PCR method. Four new KRAS and BRAF mutation detection assays were developed and their performance was demonstrated by testing a large number of replicates, utilizing a customized PCR protocol. Highly efficient and specific mutant amplification in conjunction with selective wild-type suppression by the T-blocker concept enabled 0.1% detection sensitivity using the intercalating dye-based qPCR chemistry instead of more complex target-specific dye-labeled probes. Excellent consistency in sensitivity and specificity of the T-blocker assay concept was demonstrated.

Fluorescent reversible regulation based on photoinduced electron transfer from DNA to quantum dots and intercalation binding of DNA intercalator to DNA.

Based on the fluorescent reversible regulation, a novel sensor platform was designed for the detection of DNA intercalators utilizing the intercalation binding of DNA intercalators to DNA as an inherent exhibition and the fluorescence change of quantum dots (QDs) as an external manifestation. To prove its feasibility, acridine orange (AO) was chosen as an example of DNA intercalator. When different concentrations of herring sperm DNA (hsDNA) were added to cysteamine (CA)-capped ZnSe QDs solution, the hsDNA bound with the QDs through electrostatic interaction due to the photoinduced electron transfer from hsDNA to QDs and formed QDs-hsDNA complexes with 1:1 ratio, leading to the fluorescence quenching of the QDs; and upon addition of different concentrations of AO to the QDs-hsDNA complex system, the AO first caused the release of the hsDNA from the complexes and concomitantly bound with them through intercalation binding and formed AO-hsDNA complexes with 1:3 ratio on account of the fact that the intercalation binding constant between AO and hsDNA (1.932 × 105 L/mol) was greater than the electrostatic interaction constant between QDs and hsDNA (7.874 × 104 L/mol), resulting in the fluorescence recovery of the QDs. Therefore, the detection of AO could be achieved through the relationship between the fluorescence recovery yield of the QDs and the concentration of AO added. The results illustrated that the fluorescence recovery yield of the QDs-hsDNA system was linearly dependent to the concentration of AO in the range of 5.0-75.0 × 10-5 mol/L with a detection limit (3σ/K) of 1.5 × 10-5 mol/L. This dual-directional fluorescent regulation provided a novel method for the detection of DNA intercalators such as polycyclic aromatic hydrocarbons and drugs interfering with DNA-synthesis and possessed some potential applications in the investigation of the interactions between DNA intercalators and DNA.

Magnetoresistive sensors for measurements of DNA hybridization kinetics - effect of TINA modifications.

We present the use of magnetoresistive sensors integrated in a microfluidic system for real-time studies of the hybridization kinetics of DNA labeled with magnetic nanoparticles to an array of surface-tethered probes. The nanoparticles were magnetized by the magnetic field from the sensor current. A local negative reference ensured that only the specific binding signal was measured. Analysis of the real-time hybridization using a two-compartment model yielded both the association and dissociation constants kon, and koff. The effect of probe modifications with ortho-Twisted Intercalating Nucleic Acid (TINA) was studied. Such modifications have been demonstrated to increase the melting temperature of DNA hybrids in solution and are also relevant for surface-based DNA sensing. Kinetic data for DNA probes with no TINA modification or with TINA modifications at the 5' end (1 × TINA) or at both the 5' and 3' ends (2 × TINA) were compared. TINA modifications were found to provide a relative decrease of koff by a factor of 6-20 at temperatures from 57.5 °C to 60 °C. The values of kon were generally in the range between 0.5-2 × 105 M-1s-1 and showed lower values for the unmodified probe than for the TINA modified probes. The observations correlated well with measured melting temperatures of the DNA hybrids.

Kinetics of intercalation of fluorescent probes in magnesium-aluminium layered double hydroxide within a multiscale reaction-diffusion framework.

We report the synthesis of magnesium-aluminium layered double hydroxide (LDH) using a reaction-diffusion framework (RDF) that exploits the multiscale coupling of molecular diffusion with chemical reactions, nucleation and growth of crystals. In an RDF, the hydroxide anions are allowed to diffuse into an organic gel matrix containing the salt mixture needed for the precipitation of the LDH. The chemical structure and composition of the synthesized magnesium-aluminium LDHs are determined using powder X-ray diffraction (PXRD), thermo-gravimetric analysis, differential scanning calorimetry, solid-state nuclear magnetic resonance (SSNMR), Fourier transform infrared and energy dispersive X-ray spectroscopy. This novel technique also allows the investigation of the mechanism of intercalation of some fluorescent probes, such as the neutral three-dimensional rhodamine B (RhB) and the negatively charged two-dimensional 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS), using in situ steady-state fluorescence spectroscopy. The incorporation of these organic dyes inside the interlayer region of the LDH is confirmed via fluorescence microscopy, solid-state lifetime, SSNMR and PXRD. The activation energies of intercalation of the corresponding molecules (RhB and HPTS) are computed and exhibit dependence on the geometry of the involved probe (two or three dimensions), the charge of the fluorescent molecule (anionic, cationic or neutral) and the cationic ratio of the corresponding LDH.This article is part of the themed issue 'Multiscale modelling at the physics-chemistry-biology interface'.

Development of propidium iodide as a fluorescence probe for the on-line screening of non-specific DNA-intercalators in Fufang Banbianlian Injection.

Fufang Banbianlian Injection (FBI) has been widely used as an anti-inflammatory and anti-tumor prescription. To understand the relationships between its bioactive ingredients and pharmacological efficacies, our previous study has been successfully identified some DNA-binding compounds in FBI using an established on-line screening system, in which 4',6-diamidino-2-phenylindole (DAPI) was developed as a probe. However, DAPI can be only used to screen ATT-specific DNA minor groove binders, leaving the potential active intercalators unknown in FBI. As a continuation of our studies on FBI, here we present a sensitive analytical method for rapid identification and evaluation of DNA-intercalators using propidium iodide (PI) as a fluorescent probe. We have firstly established the technique of high-performance liquid chromatography-diode-array detector-multistage mass spectrometry-deoxyribonucleic acid-propidium iodide-fluorescence detector (HPLC-DAD-MS(n)-DNA-PI-FLD) system. As a result, 38 of 58 previously identified compounds in FBI were DNA-intercalation active. Interestingly, all previously reported DNA-binders also showed intercalative activities, suggesting they are dual-mode DNA-binders. Quantitative study showed that flavonoid glycosides and chlorogenic acids were the main active compounds in FBI, and displayed similar DNA-binding ability using either DAPI or PI. In addition, 13 active compounds were used to establish the structure-activity relationships. In this study, PI was developed into an on-line method for identifying DNA-intercalators for the first time, and thus it will be a useful high-throughput screening technique for other related samples.

Pharmacogenomic study using bio- and nanobioelectrochemistry: Drug-DNA interaction.

Small molecules that bind genomic DNA have proven that they can be effective anticancer, antibiotic and antiviral therapeutic agents that affect the well-being of millions of people worldwide. Drug-DNA interaction affects DNA replication and division; causes strand breaks, and mutations. Therefore, the investigation of drug-DNA interaction is needed to understand the mechanism of drug action as well as in designing DNA-targeted drugs. On the other hand, the interaction between DNA and drugs can cause chemical and conformational modifications and, thus, variation of the electrochemical properties of nucleobases. For this purpose, electrochemical methods/biosensors can be used toward detection of drug-DNA interactions. The present paper reviews the drug-DNA interactions, their types and applications of electrochemical techniques used to study interactions between DNA and drugs or small ligand molecules that are potentially of pharmaceutical interest. The results are used to determine drug binding sites and sequence preference, as well as conformational changes due to drug-DNA interactions. Also, the intention of this review is to give an overview of the present state of the drug-DNA interaction cognition. The applications of electrochemical techniques for investigation of drug-DNA interaction were reviewed and we have discussed the type of qualitative or quantitative information that can be obtained from the use of each technique.

Quantitative Measurement of Cationic Polymer Vector and Polymer-pDNA Polyplex Intercalation into the Cell Plasma Membrane.

Cationic gene delivery agents (vectors) are important for delivering nucleotides, but are also responsible for cytotoxicity. Cationic polymers (L-PEI, jetPEI, and G5 PAMAM) at 1× to 100× the concentrations required for translational activity (protein expression) induced the same increase in plasma membrane current of HEK 293A cells (30-50 nA) as measured by whole cell patch-clamp. This indicates saturation of the cell membrane by the cationic polymers. The increased currents induced by the polymers are not reversible for over 15 min. Irreversibility on this time scale is consistent with a polymer-supported pore or carpet model and indicates that the cell is unable to clear the polymer from the membrane. For polyplexes, although the charge concentration was the same (at N/P ratio of 10:1), G5 PAMAM and jetPEI polyplexes induced a much larger current increase (40-50 nA) than L-PEI polyplexes (<20 nA). Both free cationic lipid and lipid polyplexes induced a lower increase in current than cationic polymers (<20 nA). To quantify the membrane bound material, partition constants were measured for both free vectors and polyplexes into the HEK 293A cell membrane using a dye influx assay. The partition constants of free vectors increased with charge density of the vectors. Polyplex partition constants did not show such a trend. The long lasting cell plasma permeability induced by exposure to the polymer vectors or the polyplexes provides a plausible mechanism for the toxicity and inflammatory response induced by exposure to these materials.

Sodium montmorillonite/amine-containing drugs complexes: new insights on intercalated drugs arrangement into layered carrier material.

Layered drug delivery carriers are current targets of nanotechnology studies since they are able to accommodate pharmacologically active substances and are effective at modulating drug release. Sodium montmorillonite (Na-MMT) is a clay that has suitable properties for developing new pharmaceutical materials due to its high degree of surface area and high capacity for cation exchange. Therefore Na-MMT is a versatile material for the preparation of new drug delivery systems, especially for slow release of protonable drugs. Herein, we describe the intercalation of several amine-containing drugs with Na-MMT so we can derive a better understanding of how these drugs molecules interact with and distribute throughout the Na-MMT interlayer space. Therefore, for this purpose nine sodium montmorillonite/amine-containing drugs complexes (Na-MMT/drug) were prepared and characterized. In addition, the physicochemical properties of the drugs molecules in combination with different experimental conditions were assessed to determine how these factors influenced experimental outcomes (e.g. increase of the interlayer spacing versus drugs arrangement and orientation). We also performed a molecular modeling study of these amine-containing drugs associated with different Na-MMT/drug complex models to analyze the orientation and arrangement of the drugs molecules in the complexes studied. Six amine-containing drugs (rivastigmine, doxazosin, 5-fluorouracil, chlorhexidine, dapsone, nystatin) were found to successfully intercalate Na-MMT. These findings provide important insights on the interlayer aspect of the molecular systems formed and may contribute to produce more efficient drug delivery nanosystems.

Luminescent Ir(III) complex exclusively made of polypyridine ligands capable of intercalating into calf-thymus DNA.

Efficient intercalation of a luminescent Ir(III) complex exclusively made of polypyridine ligands in natural and synthetic biopolymers is reported for the first time. The emission of the complex is largely enhanced in the presence of [poly(dA-dT)(2)] and strongly quenched in the presence of [poly(dG-dC)(2)]. By comparing the emission decays in DNA and in synthetic polynucleotides, it is proposed that the emission quenching of the title compound by guanine residues in DNA is no longer effective over a distance of four dA-dT base pairs.

Polyfluorinated bipyridine cisplatins manipulate cytotoxicity through the induction of S-G2/M arrest and partial intercalation mechanism.

A series of polyfluorinated bipyridine cisplatins 2-6 were prepared, characterized, and evaluated for their in vitro cytotoxicities against a panel of human cancer cell lines, MCF7 (breast adenocarcinoma), MDA-MB-231 (breast adenocarcinoma) and A549 (lung adenocarcinoma). The results show that a correlation between the relative order of lipophilicity of complexes 2-4 and their cytotoxicity is established by following the trend: 4>2>3. Complex 4, which is the most active compound in the series, was found to be a more effective and selective anticancer agent than cisplatin. Complex 4 inhibited cancer cell proliferation by partial intercalation to DNA, which subsequently resulted in induction of S-G2/M arrest and apoptosis.

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.

[Fluorescence of acridine orange in the composition of blood and tissues].

The interaction of acridine orange with blood albumins and tissue cells from different organs of white mouse has been studied by the spectral luminescence method. It was shown that acridine orange, by penetrating cells or orangelles, is able to intercalate between base pairs in the DNA molecule. It was found that the application of acridine orange as a fluorescent probe can influence the metabolic activity of organs.

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.

Cytotoxicity, cellular localisation and biomolecular interaction of non-covalent metallo-intercalators with appended sex hormone steroid vectors.

A range of terpyridine platinum(II) metallo-intercalators with bioactive steroids attached has been created with the aim of localizing cytotoxic drugs. Complexes where the steroid does not interfere with access to the terpyridine are shown to retain potent cytotoxicity and show certain selectivity towards their natural receptors. Because the intercalation of the terpyridine moiety between the bases of the DNA is the origin of the biological activity, a dramatic decrease of the activity is observed when the access to the terpyridine unit is hindered by the steroidal unit.

Colorimetric recognition of DNA intercalators with unmodified gold nanoparticles.

A colorimetric approach to recognize the binding mode between DNA and molecules was developed, which was based upon the electrostatic interaction between DNA and gold colloids.

High-sensitive analysis of DNA fragments by capillary gel electrophoresis using transient isotachophoresis preconcentration and fluorescence detection.

In this report aimed on further development of a high-sensitivity capillary gel electrophoresis (CGE) method for analysis of DNA fragments, we firstly explored online transient isotachophoresis (tITP) preconcentration combined with fluorescence detection (FD). The fluorescence signal (excitation: 488 nm; emission: 590 nm) was generated using the intercalating dye of ethidium bromide (EB). It was found when the leading electrolyte (LE) was injected behind the sample zone, such a special tITP mode has significant advantages to solve the bubble formation issue and to improve the analytical performance stability. Two standard DNA samples, a 50 bp DNA step ladder and the phiX174/HaeIII digest, were used to evaluate the qualitative and quantitative abilities of the tITP-FD approach. A highly diluted sample (10,000-fold in the water, e.g. the phiX174/HaeIII digest diluted from 500 microg/ml to the 50 ng/ml level) was enriched and detected; the LOD was down to 0.09 ng/ml for the 72 bp fragment, apparently improved more than 1000-fold in comparison with UV detection. Although the RSD of peak areas (n=3) was around 15.5% for the sample was electrokinetically injected, good linearity of peak area response showed that the proposed method is suitable for quantitative analysis.

Quantitation of human papillomavirus type 16 E6 oncogene sequences by real-time or quantitative PCR with EvaGreen.

Quantitation of E6 oncogene sequences of the human papillomavirus type 16 by real-time or quantitative PCR (qPCR) is used to determine the viral load, which correlates with the degree of the cervical neoplastic lesions. In the presence of EvaGreen, a new DNA intercalating fluorochrome, we obtained consistent and reproducible qPCR amplification curves and thermal denaturation profiles identical to those of the authentic E6-HPV16 (human papillomavirus 16) genome from the amplification products derived from a construct carrying the E6-HPV16 oncogene. E6-HPV16 quantitation in the presence of EvaGreen, therefore, is reproducible and specific and may be used to determine HPV16 viral load.