Newin vitro fluorimetric microtitration assays for toxicological screening of drugs

Flow cytometry has been widely used to quantify fluorescent probes in cell culture. However, FCM is not adapted to toxicological screenings due to the cost, the length and the poor reproducibility of this technique. Moreover, several multicenter studies have preferred microtitration methodologies for drug screening. A new fluorimetric technology has been designed that is sensitive and adapted to direct screening in 96-well microplates. This fluorimeter uses cold light technology (CLF) with chemical and physical modifications of the lighting system (Rat et al., 1995). CLF allows reading of UV, visible and near infrared fluorescence by increasing light energy (from 1000 to 2300 lumens) and reducing the calorific part of light (IR>900 nm, Joule effect). It induces a decrease in background and a 500- to 1000-fold improvement of detection limit of probes in comparison with classical fluorimeters and permits detection of pg/ml to fg/ml. CLF allows easy evaluation of cell injury induced by physical agents (UVA) or chemical toxins (CCl₄). Four biological endpoints for cytotoxicity evaluation have been tested with several probes: proliferation (H33258); viability (fluorescent Neutral Red); cell-cell adhesion (calcein-AM); and mitochondrial metabolic effects (Rhodamine 123). Rh 123 assay appeared more sensitive than fluorimetric or photometric detection of Neutral Red assay. Cold light fluorimetry (CLF) permits direct detection of low concentrations of probes (pg/ml to fg/ml). CLF is shown to improve classical cytotoxicity assays and, owing to its adaptability to microtitration (in 6-, 12- or 96-well plates and in Petri dishes), it is thus a promising alternative to flow cytometry for drug cytotoxicity screening.Abbreviations CLF cold light fluorimetry - FCM flow cytometry - H33258 Hoechst 33258 - IR infrared - NIR near infrared - UV ultraviolet - MTT tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide) - Rh123 Rhodamine 123     

Metal-enhanced PicoGreen fluorescence: Application for double-stranded DNA quantification

PicoGreen (PG) is a fluorescent probe for both double-stranded DNA (dsDNA) detection and quantification based on its ability to form a luminescent complex with dsDNA as compared with the free dye in solution. To expand the sensitivity of PG detection, we have studied the spectral properties of PG, both free and in complex with DNA in solution, when the fluorophore is in proximity to silver nanoparticles. We show that for a broad range of PG concentrations (20 pM-3.5 μM), it does not form dimers/oligomers and it exists in a monomeric state. On binding to DNA in the absence of silver, PG fluorescence increases approximately 1100-fold. Deposition of PG/DNA complex onto silver island films (SiFs) increases fluorescence approximately 7-fold due to the metal-enhanced fluorescence (MEF) effect, yielding fluorescence enhancement of 7700-fold as compared with the free dye on glass. In contrast to PG in complex with DNA, the free dye on SiFs demonstrates a decrease in brightness approximately 5-fold. Therefore, the total enhancement of PG on binding to DNA on silver reaches a value of approximately 38,000 as compared with free PG on SiFs. Consequently, the metal-enhanced detection of PG fluorescence is likely to find important utility for amplified dsDNA quantification.     

Enumeration of 16S rDNA of Desulfotomaculum lineage 1 in rice field soil by real-time PCR with SybrGreen detection

Real-time PCR is a new and highly sensitive method for the quantification of microbial organisms in environmental samples. This work was conducted to evaluate real-time PCR with SybrGreen (SG) detection as quantification method for Desulfotomaculum lineage 1 organisms in samples of rice field soil. The method was optimized in several parameters like SG concentration. These allowed quantitative PCR with different primer combinations yielding PCR products with lengths up to 1066 bp and with sensitivities of 10(2) targets for all assays. The detection limit in environmental DNA extracts (rice bulk soil and rice roots) was 10(6) targets per gram dry weight according to the dilution of the DNA extracts necessary to overcome PCR inhibition of humic substances. A verification, that the fluorescence increase was due to specific PCR products, was done by agarose gel electrophoresis since melting curve analysis of the PCR products did not show a distinct peak in the first derivative, when the environmental DNA extracts were used in PCR. Amplification with a primer combination specific for Desulfotomaculum lineage 1 organisms showed an abundance of this group of approximately 2% and 0.5% of the eubacterial 16S rDNA targets in rice bulk soil and rice root samples, respectively. Approximately half of this number was obtained in both habitats with a PCR assay specific for a Desulfotomaculum sequence cluster obtained previously from rice field soil.     

DNA condensation monitoring after interaction with hoechst 33258 by atomic force microscopy and fluorescence spectroscopy

DNA condensation was only observed after the addition of Hoechst 33258 (H33258) among various types of DNA binding molecules. The morphological structural change of DNA was found to depend on the H33258 concentration. On comparison of fluorescence spectrum measurements with AFM observation, it was found that fluorescence quenching of DNA-H33258 complexes occurred after DNA condensation. Additionally, we showed that DNA condensation by H33258 was independent of sequence selectivity or binding style using two types of polynucleotides, i.e. poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC). Moreover, it was concluded that the condensation was caused by a strong hydrophobic interaction, because the dissolution of condensed DNA into its native form on dimethyl sulfoxide (DMSO) treatment was observed. This study is the first report, which defines the DNA condensation mechanism of H33258, showing the correlation between the single molecule scale morphology seen on AFM observation and the bulky scale morphology observed on fluorescence spectroscopy.     

Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications

The detection of double-stranded (ds) DNA by SYBR Green I (SG) is important in many molecular biology methods including gel electrophoresis, dsDNA quantification in solution and real-time PCR. Biophysical studies at defined dye/base pair ratios (dbprs) were used to determine the structure–property relationships that affect methods applying SG. These studies revealed the occurrence of intercalation, followed by surface binding at dbprs above ~0.15. Only the latter led to a significant increase in fluorescence. Studies with poly(dA) · poly(dT) and poly(dG) · poly(dC) homopolymers showed sequence-specific binding of SG. Also, salts had a marked impact on SG fluorescence. We also noted binding of SG to single-stranded (ss) DNA, although SG/ssDNA fluorescence was at least ~11-fold lower than with dsDNA. To perform these studies, we determined the structure of SG by mass spectrometry and NMR analysis to be [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]. For comparison, the structure of PicoGreen (PG) was also determined and is [2-[N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]⁺. These structure–property relationships help in the design of methods that use SG, in particular dsDNA quantification in solution and real-time PCR.     

Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.     

A peptidyl derivative of [3H]aniline as a sensitive, stable, protease substrate.

A peptidyl derivative of [³H]aniline, Gly-Gly-Arg-[³H]anilide, can be used as a substrate in a convenient and sensitive assay procedure for trypsin, urokinase, and plasminogen activator from transformed cells. The extent of hydrolysis can be determined simply by selective extraction of the product [³H]aniline into an organic phase containing a scintillant. (The uncleaved peptide is not appreciably soluble in this phase and is not counted.) The reaction is of comparable sensitivity to fluorimetric assays, but has the advantage that no cleanup of the biological sample is required, since it is far less subject to interference from fluorescence quenching. Other peptidyl anilides should be useful for assaying proteolytic enzymes with widely varying specificities.     

A simple and sensitive DNA assay for plant extracts

A sensitive fluorimetric method was developed for the quantitative determination of DNA in plant (Zea mays L. and Medicago sativa L.) extracts. This method takes advantage of the specific increase in fluorescence intensity of the complex of DNA and the dye 4′,6′-diamidino-2-phenylindole (DAPI). Recovery of DNA and dissociation of histones from DNA were maximized by the addition of 2.0 molar NaCl to the homogenates. Treatment of the homogenate with chloroform to remove pigments and proteins decreased the quenching of fluorescence of the DAPI-DNA complex. The fluorescence intensity of RNA with DAPI was less than 2% of that produced by an equivalent weight of DNA. Comparisons were made between this fluorimetric DNA method and the commonly used diphenylamine assay for DNA. The diphenylamine DNA assay was more timeconsuming, less sensitive, and consistently resulted in lower estimates of DNA concentrations than did the fluorimetric DNA assay.     

Validation of a modified fluorimetric assay for the screening of trichomonacidal drugs

A fluorimetric microassay that uses a redox dye to determine the viability of the flagellate Trichomonas vaginalis has been optimised to provide a more sensitive method to evaluate potential trichomonacidal compounds. Resazurin has been used in recent years to test drugs against different parasites, including trichomonadid protozoa; however, the reproducibility of these resazurin-based methods in our laboratory has been limited because the flagellate culture medium spontaneously reduces the resazurin. The objective of this work was to refine the fluorimetric microassay method previously developed by other research groups to reduce the fluorescence background generated by the media and increase the sensitivity of the screening assay. The experimental conditions, time of incubation, resazurin concentration and media used in the microtitre plates were adjusted. Different drug sensitivity studies against T. vaginalis were developed using the 5-nitroimidazole reference drugs, new 5-nitroindazolinones and 5-nitroindazole synthetic derivatives. Haemocytometer count results were compared with the resazurin assay using a 10% solution of 3 mM resazurin dissolved in phosphate buffered saline with glucose (1 mg/mL). The fluorimetric assay and the haemocytometer counts resulted in similar percentages of trichomonacidal activity in all the experiments, demonstrating that the fluorimetric microtitre assay has the necessary accuracy for high-throughput screening of new drugs against T. vaginalis.     

A modified fluorimetric neutral filter elution method for analyzing radiation-induced double strand break and repair

Neutral filter elution assay is one of the methods used for detection of DNA double strand breaks (DSBs). However, it is laborious, expensive, and hazardous (radiolabeled precursors for DSB detection and scintillation counter for quantification), making it a less preferred method for DSB detection. In the present study, an attempt was made to improve the existing neutral filter elution assay by making use of fluorescent dye (PicoGreen) and microfiltration assembly for eluting the fragmented DNA, thereby reducing the cost and time required for the assay. We studied the effect of dye dilution, pH conditions, and cell number as a part of method standardization. X-ray dose–response and repair kinetics in lymphocytes as well as cell lines were studied for validating the sensitivity of the assay. A linear dose–response relationship for DSBs was observed at a cell number of 4 × 10⁵ cells, a dye dilution of 500-fold, and at pH 10. Repair kinetics revealed a time-dependent repair of DSBs up to 360 min of posttreatment, indicating its usefulness in DSB repair studies. In conclusion, the present modified method is more efficient (in terms of cell number), cost effective, less time-consuming, and less hazardous compared to the existing method.     

Comparison of folate quantification in foods by high-performance liquid chromatography-fluorescence detection to that by stable isotope dilution assays using high-performance liquid chromatography-tandem mass spectrometry

A comparison study on folate quantitation was carried out between the recently developed stable isotope dilution assay using liquid chromatography-tandem mass spectrometry (LC-MS-MS) and the frequently used HPLC with fluorimetric detection (LC-FD). By applying LC-MS-MS, spinach, wheat bread, beef, and blood plasma were found to contain 159.2, 19.8, 1.2, and 5.6 microg/100 g total folates, respectively, whereas the respective quantitative data obtained by LC-FD were 95.5, 16.2, 0.7, and 6.8 microg/100 g. In all samples, LC-MS-MS revealed superior selectivity and precision and circumvented the shortcomings of conventional LC techniques, i.e., ambiguous peak assignment as well as high detection limits for 5-formyltetrahydrofolate, 10-formylfolic acid, and folic acid. The affinity chromatography columns used in this study showed excellent cleanup performance and permitted detection limits as low as 0.1, 0.5, 0.1, 0.08, and 0.1 microg/100 g for tetrahydrofolate (H(4)folate), 5-methyl-H(4)folate, 5-formyl-H(4)folate, 10-formylfolate, and pteroylglutamic acid, respectively. Thus, a 10-fold higher sensitivity compared to solid-phase anion-exchange cartridges was achieved. However, affinity chromatography columns revealed a significantly higher affinity toward the natural vitamers than to the racemic isotopomeric standards, which has to be considered when applying the latter in stable isotope dilution assays.     

Apparent inhibition of apoptosis by polyamines and aminothiols in DNA fragmentation assays is artifactual

We report that, in commonly used DNA fragmentation assays, polyamines and the radioprotective aminothiol WR1065 artifactually depress the degree of spontaneous or induced cellular apoptosis in two distinct ways. Firstly, in assays utilizing Hoechst 33258 dye to measure apoptotic DNA, both amines quench the fluorescence of low affinity dye/DNA binding resulting in preferential underestimation of DNA in the apoptotic DNA fraction and a resultant underestimation of the extent of DNA fragmentation. Secondly, these amines can cause aggregation and condensation of apoptotic DNA, causing anomalous sedimentation under conditions universally employed to separate apoptotic from intact DNA in cell lysates. This anomalous sedimentation of apoptotic DNA leads to underestimation of fragmentation in fluorescence assays as well as in agarose gel assays. We demonstrate that manipulation of the ionic strength of the lysis buffer or lowering the dye concentration ameliorates the effects of dye quenching in the Hoechst assay. Alternatively, this effect is alleviated by substituting DAPI for Hoechst in this assay. Inclusion of a polyanion to the lysis buffer antagonizes the condensation and anomalous sedimentation of apoptotic DNA observed regardless of which dye is used in the assay. These studies call into question the validity of previously reported studies suggesting that polyamines and the radioprotective aminothiol, WR1065, inherently suppress the apoptotic process and underline the need to consider alternative endpoints of apoptosis such as morphology in order to assess effects on cellular apoptosis of exogenously added agents, particularly di- or polycations.     

A solid-phase immunoassay for the binding of cartilage proteoglycan to hyaluronic acid

A solid-phase assay for detecting the binding of cartilage proteoglycan (PG) to hyaluronic acid (HA) is described. In the assay, HA is immobilized on protamine-treated microtiter wells, the wells are incubated with PG monomer and antibody to PG monomer, and then an ELISA system is used to detect binding of the PG to HA. The specificity of the assay is indicated by the failure to detect PG binding to chondroitin sulfate or albumin-coated microtiter wells, the absence of binding with tryptic fragments of PG monomer other than the HA-binding segment, the loss of binding after reduction and alkylation of PG monomer, and the inhibition of binding by preincubation of PG monomer with small amounts of HA. In contrast to the HA-PG interaction in solution, hyaluronidase digestion of HA does not affect its ability to inhibit the reaction of PG monomer with immobilized HA. The microtiter well-based assay appears to be a rapid, simple, and potentially versatile method for studying interactions with HA.     

Increasing the analytical sensitivity by oligonucleotides modified with para- and ortho-twisted intercalating nucleic acids--TINA

The sensitivity and specificity of clinical diagnostic assays using DNA hybridization techniques are limited by the dissociation of double-stranded DNA (dsDNA) antiparallel duplex helices. This situation can be improved by addition of DNA stabilizing molecules such as nucleic acid intercalators. Here, we report the synthesis of a novel ortho-Twisted Intercalating Nucleic Acid (TINA) amidite utilizing the phosphoramidite approach, and examine the stabilizing effect of ortho- and para-TINA molecules in antiparallel DNA duplex formation. In a thermal stability assay, ortho- and para-TINA molecules increased the melting point (Tm) of Watson-Crick based antiparallel DNA duplexes. The increase in Tm was greatest when the intercalators were placed at the 5' and 3' termini (preferable) or, if placed internally, for each half or whole helix turn. Terminally positioned TINA molecules improved analytical sensitivity in a DNA hybridization capture assay targeting the Escherichia coli rrs gene. The corresponding sequence from the Pseudomonas aeruginosa rrs gene was used as cross-reactivity control. At 150 mM ionic strength, analytical sensitivity was improved 27-fold by addition of ortho-TINA molecules and 7-fold by addition of para-TINA molecules (versus the unmodified DNA oligonucleotide), with a 4-fold increase retained at 1 M ionic strength. Both intercalators sustained the discrimination of mismatches in the dsDNA (indicated by ΔTm), unless placed directly adjacent to the mismatch--in which case they partly concealed ΔTm (most pronounced for para-TINA molecules). We anticipate that the presented rules for placement of TINA molecules will be broadly applicable in hybridization capture assays and target amplification systems.     

Multi-spectroscopic and molecular docking studies on the interaction of neotame with calf thymus DNA

Abstract

In this paper, we have studied the in vitro binding of neotame (NTM), an artificial sweetener, with native calf thymus DNA using different methods including spectrophotometric, spectrofluorometric, competition experiment, circular dichroism (CD), and viscosimetric techniques. From the spectrophotometric studies, the binding constant (K_b) of NTM-DNA was calculated to be 2?×?10³ M^?1. The quenching of the intrinsic fluorescence of NTM in the presence of DNA at different temperatures was also used to calculate binding constants (K_b) as well as corresponding number of binding sites (n). Moreover, the obtained results indicated that the quenching mechanism involves static quenching. By comparing the competitive fluorimetric studies with Hoechst 33258, as a known groove probe, and methylene blue, as a known intercalation probe, and iodide quenching experiments it was revealed that NTM strongly binds in the grooves of the DNA helix, which was further confirmed by CD and viscosimetric studies. In addition, a molecular docking method was employed to further investigate the binding interactions between NTM and DNA, and confirm the obtained results.     

Mutations at Arginine 276 transform human uracil-DNA glycosylase into a single-stranded DNA-specific uracil-DNA glycosylase

To investigate the role of Arginine 276 in the conserved leucine-loop of human uracil-DNA glycosylase (UNG), the effects of six R276 amino acid substitutions (C, E, H, L, W, and Y) on nucleotide flipping and enzyme conformational change were determined using transient and steady state, fluorescence-based, kinetic analysis. Relative to UNG, the mutant proteins exhibited a 2.6- to 7.7-fold reduction in affinity for a doubled-stranded oligonucleotide containing a pseudouracil residue opposite 2-aminopurine, as judged by steady-state DNA binding-base flipping assays. An anisotropy binding assay was utilized to determine the K(d) of UNG and the R276 mutants for carboxyfluorescein-labeled uracil-containing single- and double-stranded oligonucleotides; the binding affinities varied 11-fold for single-stranded uracil-DNA, and 43-fold for double-stranded uracil-DNA. Productive uracil-DNA binding was monitored by rapid quenching of UNG intrinsic protein fluorescence. Relative to UNG, the rate of intrinsic fluorescence quenching of five mutant proteins for binding double-stranded uracil-DNA was reduced approximately 50%; the R276E mutant exhibited 1% of the rate of fluorescence quenching of UNG. When reacted with single-stranded uracil-DNA, the rate of UNG fluorescence quenching increased. Moreover, the rate of fluorescence quenching for all the mutant proteins, except R276E, was slightly faster than UNG. The k(cat) of the R276 mutants was comparable to UNG on single-stranded DNA and differentially affected by NaCl; however, k(cat) on double-stranded DNA substrate was reduced 4-12-fold and decreased sharply at NaCl concentrations as low as 20 mM. Taken together, these results indicate that the effects of mutations at Arg276 were largely limited to enzyme interactions with double-stranded uracil-containing DNA, and suggested that mutations at Arg276 effectively transformed UNG into a single-stranded DNA-specific uracil-DNA glycosylase.     

Monitoring influenza virus content in vaccine production: precise assays for the quantitation of hemagglutination and neuraminidase activity

Robust and precise quantitation of influenza virus is a premise for the efficient development of vaccine production processes. In this article, revised assays for the determination of hemagglutination (HA) and neuraminidase (NA) activity are presented. Bias of traditional discontinuous HA assays and operator dependency was overcome by introduction of a regression procedure. At little effort, a continuous assay result is obtained with repeatability as good as +29%/-22% in the best case (95% confidence intervals reported). Similarly, neuraminidase activity determined in microtiter plates resulted in repeatability better than +/-20%. NA activity decreased almost linearly for pH ranging from 5.8 to 7.8 and was enhanced by the addition of Ca2+. Non-linearity of the assay (due to unspecific adsorption) was overcome by addition of BSA. Using 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid as substrate, Michaelis-Menten constants of 30 and 460 microM were determined for strains A/PR/8/34 (H1N1) and A/Equi 2/NM/1/93 (H3N8), respectively. The error introduced by approximation of Michaelis-Menten kinetics (zero and first order) was minimized by limiting substrate consumption to about 10%. Linearity of both assays was verified in dilution experiments. Applicability was demonstrated in three cases: virus propagation in mammalian cell culture, ultrafiltration and precipitation of nucleic acids.     

Microplate fluorescence assay for the quantification of double stranded DNA using SYBR Green I dye

A high-throughput, 96-well microplate fluorescence assay (MFA) was developed for DNA quantification using the double-stranded DNA-binding dye SYBR Green I. Samples mixed with SYBR Green I in the wells of a microtiter plate produced fluorescence in proportion with DNA concentration which was measured using a fluorescence plate reader. The performance characteristics of the assay were compared with spectrophotometric quantification based on ultraviolet absorption and the Hoefer DyNA Quant assay utilizing the fluorescent dye, Hoechst 33258. The MFA accurately quantified different types of DNA over a broad linear dynamic range of concentrations (0.25–2,500 pg/μl), and was not affected by a variety of contaminants in the assay mixture.     

Assay for nanogram quantities of DNA in cellular homogenates.

The DNA concentration of a crude cellular homogenate can be measured accurately in the nanogram range using the fluorescence enhancement of 4′,6-diamidino-2-phenylindole (DAPI) or bisbenzimidazole (Hoechst H 33258) complexed with DNA. A simple assay has been devised including an internal standard, which allows reliable measurement and compensates for any quenching due to cellular components or buffer. The fluorescence enhancement is highly specific for DNA; no other cell component produces significant fluorescence. The response is linear over a broad dynamic range making the measurement of unknown DNA concentrations convenient.