Synthesis of N-4-butylamine acridone and its use as fluorescent probe for ctDNA

A novel N-4-butylamine acridone (BAA), which is an acridone derivative was synthesized and characterized by IR, MS and (1)H NMR. The fluorescent characteristics of BAA was investigated in detail and used as the fluorescent probe for detection of calf thymus DNA (ctDNA). It was found that DNA was able to quench the fluorescence of BAA at 426 nm with the excitation at 254 nm. Under optimal conditions, the corresponding linear response range was from 1.0 to 20.0mg/L and the limit of detections (LOD) was 0.020 mg/L (defined as S/N=3). Moreover, the interaction between BAA and ctDNA was investigated by fluorescence, absorption and viscosity measurements. The results suggested that the interaction between BAA and ctDNA is groove binding in nature.     

Determination of nucleic acids based on the quenching effect on resonance light scattering of the Y(III)-1,6-bi(1'-phenyl-3'-methyl-5'-pyrazolone-4'-)hexane-dione system.

Nucleic acids can quench resonance light scattering (RLS) intensity of the Y(III)-1,6-bi(1'-phenyl-3'-methyl-5'-pyrazolone-4'-)hexane-dione(BPMPHD) complex in the pH range 5.0-5.8. Under optimal conditions, there are linear relationships between the quenching of RLS and the concentration of nucleic acids in the range 6.3 x 10(-8)-2.1 x 10(-5) g/mL for fish sperm DNA (fsDNA), 1.2 x 10(-8)-5.0 x 10(-5) g/mL for calf thymus DNA (ctDNA) and 6.0 x 10(-8)-2.0 x 10(-5) g/mL for yeast RNA (yRNA). The detection limits (3 s) of fsDNA, ctDNA and yRNA are 0.7 ng/mL, 3.8 ng/mL and 4.2 ng/mL, respectively.     

Spectroscopic and computational approaches to unravel the mode of binding between a isoflavone,biochanin-A and calf thymus DNA

In the present study, attempt was made to explore the interaction between biochanin-A (BioA) and calf thymus DNA (ctDNA) by employing fluorescence spectroscopy, absorption spectroscopy, circular dichroism (CD), DNA melting studies, viscosity measurements, and molecular modeling methods. A well-known fluorescence probe, acridine orange (AO) was used in the present study in order to enhance the emission intensity of weakly fluorescent ctDNA. Quenching in emission intensity of ctDNA-AO system was observed in the presence of different concentrations of BioA, suggesting that BioA has interacted with ctDNA. The hyperchromic effect observed upon the addition of BioA in the absorption spectra of ctDNA-AO without any shift in its absorption maximum revealed that BioA was bound to ctDNA through groove mode of binding. Further the groove mode of binding of BioA to ctDNA was confirmed by DNA melting studies, viscosity measurements, and molecular docking studies. The results of fluorescence measurements that were carried out at different temperature indicated that the BioA has quenched the emission intensity of ctDNA-AO through static mode of quenching mechanism. Thermodynamic parameters revealed that the BioA-ctDNA-AO system was stabilized by van der Waals forces and hydrogen bonding. The effect of binding of BioA on the conformation of ctDNA was examined by circular dichroism studies.     

The fluorescence enhancement of quercetin–nucleic acid system and the analytical application

Nucleic acid can greatly enhance the fluorescence intensity of quercetin in HMTA‐HCl (pH 5.5) buffer. The enhanced intensity is in proportion to the concentration of nucleic acids in the range 5.0 × 10^?9 to 1.0 × 10^?6 g/mL for fsDNA, 5.0 × 10^?9 to 7.0 × 10^?7 g/mL for ctDNA and 5.0 × 10^?9 to 1.0 × 10^?6 g/mL for yRNA, and their detection limits (S/N = 3) are 3.5 × 10^?9, 7.8 × 10^?10 and 2.6 × 10^?9 g/mL, respectively. In comparison with most reported fluorescent probes for the determination of nucleic acids, the proposed probe has higher sensitivity and lower toxicity. The interaction investigation indicates that quercetin binds with double‐strand DNA in groove binding mode, resulting in fluorescence enhancement of this system. Copyright © 2009 John Wiley & Sons, Ltd.     

Systematical investigation of binding interaction between novel ruthenium(II) arene complex with curcumin analogs and ctDNA

In this study, the interaction between a novel ruthenium(II) arene complex with curcumin analogs and calf thymus DNA (ctDNA) was investigated systematically by viscosity measurement, the DNA melting approach, multispectroscopic techniques and electrochemical methods. The absorption spectra of the ctDNA–drug complex showed a slight red shift and a weak hypochromic effect. The relative viscosity and melting temperature of ctDNA increased on addition of the drug. The evidence obtained from fluorescence competitive experiments indicated that the binding mode of the drug with ctDNA was intercalative. Using acridine orange (AO) as a fluorescence probe, the drug statically quenched the fluorescence of the ctDNA–AO complex, and hydrogen bonding and van der Waals interactions played vital roles in the binding interaction between the drug and ctDNA. The influences of ionic strength, chemical denaturants and pH on the binding interaction were also investigated. Circular dichroism and Fourier transform infrared spectra suggested that this drug might bond with the G–C base pairs of ctDNA and the right‐handed B‐form helicity of ctDNA remained after drug binding. The intercalative binding between the drug and ctDNA was further investigated using electrochemical techniques. All these results suggested that the biological activity of ctDNA was affected by ruthenium(II) arene complex with curcumin analogs. Copyright © 2016 John Wiley & Sons, Ltd.     

Spectrofluorimetric determination of heparin using a tetracycline-europium probe

A new spectrofluorimetric method was developed for the determination of trace amounts of heparin (Hep). Using tetracycline (TC)-europium ion (Eu3+) as a fluorescent probe, in the buffer solution at pH 8.8, Hep can remarkably enhance the fluorescence intensity of the TC-Eu3+ complex at lambda=612 nm and the enhanced fluorescence intensity of Eu3+ is in proportion to the concentration of Hep. Optimal conditions for the determination of Hep were also investigated. The linear range and detection limit for the determination of Hep were 0.02 to 1.6 microg/ml and 4.45 ng/ml, respectively. This method is simple, practical, and relatively free of interference from coexisting substances, and it can be applied successfully to assess Hep in biological samples. By the Rosenthal graphic method, the association constant and binding numbers of Hep with the probe were 4.46 x 10(4) L/mol and 16.2, respectively. Moreover, the enhancement mechanism of the fluorescence intensity in the TC-Eu3+ system, the TC-Eu(3+)-Hep system, and the TC-Eu(3+)-Hep-CTMAB system is also discussed.     

The sensitive determination of nucleic acids using a fluorescence-quenching method.

Experiments indicated that nucleic acids can quench the fluorescence of the Eu3+ -2-thenoyltrifluoroacetone (TTA)-1,10-phenanthroline (Phen) system. Based on this, a sensitive method for the determination of nucleic acids was proposed. The experiments indicated that under the optimum conditions, the quenched fluorescence intensity was in proportion to the concentration of nucleic acids in the range 1.0 x 10(-11)-1.0 x 10(-6) g/mL for yeast RNA (yRNA), 5.0 x 10(-11)-5.0 x 10(-7) g/mL for fish sperm (fsDNA) and 1.0 x 10(-10)-1.5 x 10(-6) g/mL for calf thymus DNA (ctDNA). Their detection limits were 3.0 x 10(-12), 4.0 x 10(-12) and 5.0 x 10(-11) g/mL, respectively. Therefore, the proposed method is one of the most sensitive methods available. The interaction between nucleic acids and Eu3+ -TTA-Phen is also discussed.     

Reinterpretation of fluorescence of terbium ion-DNA complexes

Terbium (Tb3+) fluorescence was used to investigate local non-denaturation perturbations of double-helical DNA structure induced in this nucleic acid by various physical and chemical agents. It has been shown that the interaction of Tb3+ with DNA into which single-strand or double-strand breaks have been introduced by DNase I or by low doses of ionizing radiation does not influence the fluorescence of the lanthanide cation. On the other hand, interaction of terbium with DNA modified by the antitumour drug cis-diamminedichloroplatinum(II) at low levels of binding and by low doses of ultraviolet radiation (wavelength 254 nm) has been shown to result in substantial enhancement of the fluorescence of this cation. It has been proposed that the terbium fluorescent probe can also be exploited successfully for the purpose of analysing the guanine bases present in distorted double-stranded regions of DNA, in which only the vertical stacking of the base-pairs is altered.     

Curcumin-embedded DBPC liposomes coated with chitosan layer as a fluorescence nanosensor for the selective detection of ribonucleic acid

One of the limitations of fluorescence probe molecules during biomedical estimation is their lack of ability to selectively determine the targeted species. To overcome this there have been various approaches that involve attaching a functional group or aptamers to the fluorescence probe. However, encapsulating probe molecules in a matrix using nanotechnology can be a viable and easier method. Curcumin (Cur) as a fluorescence marker cannot distinguish DNA and RNA. This research reports a novel selective approach involving the use of nanocapsules composed of liposomal curcumin coated with chitosan for the selective detection of RNA molecules using a fluorescence method. The increase in RNA concentration enhanced the electrostatic interaction between the negatively charge surface of RNA and the positively charged nanocapsule, which was further verified by zeta potential measurement. This method had a low limit of detection (36 ng/ml) and higher linear dynamic ranges compared with other studies found in the literature. Moreover, the method was not affected by DNA and was selective for the detection of RNA molecules for which the site of interaction was confined only to uracil. The selectivity for RNA molecules towards other analogues species was also examined and recovery range found was between 99 and 100.33%.     

Synthesis of a new Ni-phenanthroline complex and its application as an electrochemical probe for detection of nucleic acid

A new DNA sensor using a nickel(II) phenanthroline complex ([Ni(phen)(2)PHPIP]·2ClO(4)) as the electrochemical probe was developed. The sensor is very sensitive and selective for calf thymus DNA (ctDNA) detection in aqueous medium. The Ni-phenanthroline probe was synthesized by a two-step preparation using p-hydroxy-phenylimidazo-1,10-phenanthroline (PHPIP) as the ligand and characterized with IR, UV and MS. Some interesting electrochemical properties of the Ni-complex and the interactions of the complex with ctDNA were reported. The calculated dynamics parameters of the electrode process indicate that there are obvious interactions between the probe and the ctDNA in aqueous solution. Under constant potential conditions, the redox current peak of the probe (Ni-complex) decreases obviously as the probe interacts/binds with ctDNAs. This unexpected electrochemical behavior may suggest that a new adduct through the binding of Ni-phenanthroline complex with ctDNA is formed electrochemically. By estimation, the binding ratio of the probe and ctDNA was found to be 1:1 with a binding constant β=4.29×10(5) mol L(-1) in aqueous solution at room temperature.     

Binding Interactions of Naringenin and Naringin with Calf Thymus DNA and the Role of β-Cyclodextrin in the Binding

The interaction of naringenin (Nar) and its neohesperidoside, naringin (Narn), with calf thymus deoxyribonucleic acid (ctDNA) in the absence and the presence of β-cyclodextrin (β-CD) was investigated. The interaction of Nar and Narn with β-CD/ctDNA was analyzed by using absorption, fluorescence, and molecular modeling techniques. Docking studies showed the existence of hydrogen bonding, electrostatic and phobic interaction of Nar and Narn with β-CD/DNA. 1:2 stoichiometric inclusion complexes were observed for Nar and Narn with β-CD. With the addition of ctDNA, Nar and Narn resulted into the fluorescence quenching phenomenon in the aqueous solution and β-CD solution. The binding constant K _b and the number of binding sites were found to be different for Nar and Narn bindings with DNA in aqueous and β-CD solution. The difference is attributed to the structural difference between Nar and Narn with neohesperidoside moiety present in Narn.     

Interaction of 3'-azido-3'-deamino daunorubicin with DNA: multispectroscopic and molecular modeling

In this paper, 3'-azido-3'-deamino daunorubicin (ADNR) was synthesized and the interaction of ADNR and calf thymus DNA (ctDNA) was investigated for the first time by using multi-spectroscopic techniques and molecular modeling study in vitro under simulated physiological conditions. Hypochromicity of the absorption spectra of ADNR were observed in the presence of ctDNA, and the fluorescence of ADNR was strongly quenched through static mechanism with the addition of ctDNA. Moreover, the fluorescence polarization was increased when ctDNA was added. From the experimental results, conclusion can be drawn that the binding mode of ADNR with ctDNA was an intercalative binding, and the calculated thermodynamic parameters suggested that the binding of ADNR to ctDNA was driven mainly by hydrogen bonding force. Furthermore, the results obtained from computational modeling further proved the experimental results obtained from spectroscopic investigations.     

Study on interactions of aminoglycoside antibiotics with calf thymus DNA and determination of calf thymus DNA via the resonance Rayleigh scattering technique

A simple and sensitive resonance Rayleigh scattering (RRS) spectra method was developed for the determination of calf thymus DNA (ctDNA). The enhanced RRS signals were based on the interactions between ctDNA and aminoglycoside antibiotics (AGs) including kanamycin (KANA), tobramycin (TOB), gentamicin (GEN) and neomycin (NEO) in a weakly acidic medium (pH 3.3–5.7). Parameters influencing the method were investigated. Under optimum conditions, increments in the scattering intensity (?I) were directly proportional to the concentration of ctDNA over certain ranges. The detection limit ranged from 12.2 to 16.9 ng/mL. Spectroscopic methods, including RRS spectra, absorption spectra and circular dichroism (CD) spectroscopy, coupled with thermo‐denaturation experiments were used to study the interactions, indicating that the interaction between AGs with ctDNA was electrostatic binding mode. Copyright © 2015 John Wiley & Sons, Ltd.     

Luminescent and hydrophilic LaF3–polymer nanocomposite for DNA detection

Luminescent LaF₃–Ce³⁺/Tb³⁺ nanocrystals have been successfully prepared via a simple wet chemical technique. For the next bioapplication, these nanoparticles dispersed in cyclohexane have also been functionalized with poly(St‐co‐MAA), based on a designed oil‐in‐water microemulsion system. These polymer‐coated nanospheres are water‐soluble and bioconjugable. Unlike semiconductor quantum dots, the as‐prepared lanthanum fluoride nanocrystals possess non‐size‐dependent emissions and completely stable photocycles. With functionalized LaF₃ nanospheres as fluorescence probes, a fluorescence method was developed for the rapid quantitative analysis of DNA, due to the quenching effect of fluorescence by the DNA. Under optimum conditions, the fluorescence intensity was proportional to the concentration of the introduced DNA over the range 2.5–35 µg/mL for calf thymus DNA (ctDNA) and 2.5–30 µg/mL for fish sperm DNA (fsDNA), respectively. Copyright © 2008 John Wiley & Sons, Ltd.     

Detection of salmonellas by DNA hybridization with a fluorescent alkaline phosphatase substrate.

This study evaluates a DNA hybridization assay for salmonella with AttoPhos (JBL Scientific, San Luis Obispo, CA), a fluorescent substrate for alkaline phosphatase. The probe used (50 ng/ml) was a biotinylated 600 bp fragment consisting of a tandem repeat of an insertion sequence (IS200) found in most Salmonella spp. evaluated. The hybridization was carried out at 65 degrees C for 2 h without prior prehybridization and hybrids were detected by the addition of a streptavidin-alkaline phosphatase conjugate. Circles (5 mm) were cut from the membrane and placed in a cuvette containing 1 ml of 1 mmol/l AttoPhos. The reaction was evaluated after 30 min at 37 degrees C with a fluorometer with an excitation wavelength of 440 nm and an emission wavelength of 550 nm. The sensitivity of the probe was estimated to be 10,000 copies of target DNA or 5 x 10(-20) mol of DNA. All 74 salmonella strains tested reacted with the probe but none of the 98 heterologous species tested gave positive results. The results of this study indicate that our assay method, which employs a biotinylated tandem repeat of IS200 and AttoPhos, is a specific and highly sensitive quantitative method for the detection of salmonellas.     

Label-less fluorescence-based method to detect hybridization with applications to DNA micro-array

By coupling scattered light from DNA to excite fluorescence in a polymer, we describe a quantitative, label-free assay for DNA hybridization detection. Since light scattering is intrinsically proportional to number of molecules, the change in (scattering coupled) fluorescence is highly linear with respect to percent binding of single stranded DNA (ssDNA) target with the immobilized ssDNA probes. The coupling is achieved by immobilizing ssDNA on a fluorescent polymer film at optimum thickness in nanoscale. The fluorescence from the underlining polymer increases due to proportionate increase in scattering from double stranded DNA (dsDNA) (i.e., probe-target binding) compared to ssDNA (i.e., probe). Because the scattering is proportional to fourth power of refractive index, the detection of binding is an order of magnitude more sensitive compared to other label-free optical methods, such as, reflectivity, interference, ellipsometry and surface-plasmon resonance. Remarkably, polystyrene film of optimum thickness 30 nm is the best fluorescent agent since its excitation wavelength matches (within 5 nm) with wavelength for the maximum refractive index difference between ssDNA and dsDNA. A quantitative model (with no fitting parameters) explains the observations. Potential dynamic range is 1 in 10(4) at signal-to-noise ratio of 3:1.     

DNA-cholesterol complex studied by fluorescent probes

DNA interaction with cholesterol at various lipid concentrations has been investigated by the fluorescent probes method. It has been shown that the intensity of acridine orange fluorescence in the DNA-cholesterol complex decreases at 24 micrograms/ml cholesterol and at 45 micrograms/ml it increases. The number of binding sites and the degree of polarization of fluorescence change simultaneously. Binary mechanism of cholesterol binding with DNA has been suggested: surface binding takes place at low concentrations, intercalation--at high lipid concentrations.     

High performance liquid chromatographic determination of topiramate in human serum using UV detection

Topiramate has no ultraviolet, visible or fluorescence absorption. Analysis of the drug in human serum has been reported by high performance liquid chromatography (HPLC) with either mass detector or fluorescence detection after precolumn derivatization using 9-fluorenylmethyl chloroformate as fluorescent labeling agent. This study was aimed to validate derivatization and analysis of topiramate in human serum with HPLC using UV detection. The drug was extracted from human serum by liquid-liquid extraction and subjected to derivatization with 9-fluorenylmethyl chloroformate. Analysis was performed on a phenyl column using of spectrophotometer detection operated at wavelength of 264 nm. A mixture of phosphate buffer (0.05M) containing triethylamine (1 ml/l, v/v; pH 2.3) and methanol (28:72, v/v) at a flow rate of 2.5 ml/min was used as mobile phase. No interference was found with endogenous substances. Validity of the method was studied and the method was precise and accurate with a linearity range from 40 ng/ml to 40 microg/ml. The limit of quantification was 40 ng/ml of serum. The correlation coefficient between HPLC methods using fluorescence and UV detections was studied and found to be 0.992.     

Capecitabine as a minor groove binder of DNA: molecular docking,molecular dynamics,and multi-spectroscopic studies

The interaction mechanism and binding mode of capecitabine with ctDNA was extensively investigated using docking and molecular dynamics simulations, fluorescence and circular dichroism (CD) spectroscopy, DNA thermal denaturation studies, and viscosity measurements. The possible binding mode and acting forces on the combination between capecitabine and DNA had been predicted through molecular simulation. Results indicated that capecitabine could relatively locate stably in the G-C base-pairs-rich DNA minor groove by hydrogen bond and several weaker nonbonding forces. Fluorescence spectroscopy and fluorescence lifetime measurements confirmed that the quenching was static caused by ground state complex formation. This phenomenon indicated the formation of a complex between capecitabine and ctDNA. Fluorescence data showed that the binding constants of the complex were approximately 2 × 10⁴ M^?1. Calculated thermodynamic parameters suggested that hydrogen bond was the main force during binding, which were consistent with theoretical results. Moreover, CD spectroscopy, DNA melting studies, and viscosity measurements corroborated a groove binding mode of capecitabine with ctDNA. This binding had no effect on B-DNA conformation.     

Groove binding between ferulic acid and calf thymus DNA: spectroscopic methodology combined with chemometrics and molecular docking studies

Abstract

Ferulic acid (FA), a dietary phenolic acid compound, is proved to possess numerous biological activities. Hence, this study was devoted to explore the interaction between FA and calf thymus DNA (ctDNA) by UV???vis absorption, fluorescence, circular dichroism (CD) spectroscopy combined with multivariate curve resolution-alternating least-squares (MCR???ALS) and molecular docking studies. The concentration curves and the pure spectra of compositions (FA, ctDNA and FA???ctDNA complex) were obtained by MCR???ALS approach to verify and monitor the interaction of FA with ctDNA. The groove binding mode between FA and ctDNA was confirmed by the results of melting analysis, viscosity measurements, single-stranded DNA experiments, and competitive studies. The binding constant of FA???ctDNA complex was 4.87?×?10⁴ L mol^?1 at 298?K. The values of enthalpy (ΔH°) and entropy (ΔS°) changes in the interaction were ?16.24?kJ mol^?1 and 35.02?J mol^?1 K^?1, respectively, indicating that the main binding forces were hydrogen bonds and hydrophobic interactions. The result of CD spectra suggested that a decrease in right-handed helicity of ctDNA was induced by FA and the DNA conformational transition from the B-form to the A-form. The results of docking indicated that FA binding with ctDNA in the minor groove. These findings may be conducive to understand the interaction mechanism of FA with ctDNA and the pharmacological effects of FA.

Communicated by Ramaswamy H. Sarma