Quantitative Determination of DNA–Ligand Binding Using Fluorescence Spectroscopy (original) (raw)
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Journal of Chemical Education, 2007
DAPI, 4´,6-diamidino-2-phenylindole ), binds to double-stranded DNA forming a complex that fluoresces up to twenty times more than DAPI alone (1). The complex is stable for hours at room temperature and is unchanged over pH 4-11. The experiment described here measures the DAPI binding constant, K f , for calf-thymus DNA using fluorescence measurements and a modified Scatchard analysis. This experiment also allows the student to estimate the occupancy of the minor groove by calculating the number of nucleotides per bound ligand.
Nucleic Acids Research, 2003
Ensemble and single-molecule¯uorescence measurements of 2¢-(4-hydroxyphenyl)-5- [5-(4-methylpiperazine-1-yl) benzimidazo-2-yl]-benzimidazole (H-258)± calf thymus (CT) DNA complexes at various [H-258]/ [DNA bp] ratios were performed to elucidate the binding of H-258 with DNA. Upon binding to doublestranded CT DNA (CT ds DNA) at a [H-258]/[DNA bp] ratio of 0.05 the relative¯uorescence quantum yield, F f , of H-258 increases from 0.02 to 0.58. Thē uorescence decay can be ®tted almost by a monoexponential model with a lifetime of~3.6 ns. This indicates that H-258 binds almost quantitatively in the minor groove of DNA at low [H-258]/[DNA bp] ratios. With increasing [H-258]/[DNA bp] ratios, e.g. 0.15 and 0.20, the¯uorescence quantum yield of H-258 decreases to 0.28 and 0.19, respectively. Fitting of the¯uorescence decays measured for higher [H-258]/[DNA bp] ratios reveals the presence of additional shorter¯uorescence lifetime components in the range of 0.5±2.0 ns. Our results suggest that H-258 partially intercalates in G:C sequences at higher [H-258]/[DNA bp] ratios re¯ected by a lifetime component of 1.5±2 ns. In addition, stacking or adsorption of H-258 molecules on DNA occurs at higher [H-258]/[DNA bp] ratios.
Fluorescence spectroscopy and anisotropy in the analysis of DNA-protein interactions
Methods in molecular biology (Clifton, N.J.), 2009
Fluorescence spectroscopy can be used as a sensitive non-destructive technique for the characterisation of protein-DNA interactions. A comparison of the intrinsic emission spectra obtained for a protein-DNA complex and for free protein can be informative about the environment of tryptophan and tyrosine residues in the two states. Often there is quenching of the fluorescence intensity of an intrinsic emission spectrum and/or a shift in the wavelength maximum on protein binding to DNA. A step-by-step protocol describes the determination of a DNA-binding curve by measurement of the quenching of the intrinsic protein fluorescence.Fluorescence anisotropy can also be used to obtain a DNA-binding curve if the molecular size of the protein-DNA complex is sufficiently different from the free fluorescing component. Typically an extrinsic fluorophore attached to one or both 5' ends of single-stranded or duplex DNA is used, for this increases the sensitivity of measurement.Fitting of the bi...
Analytical Chemistry, 2002
Four oligonucleotides (fluorescently labeled and unlabeled 16-and 90-mer), each containing a single adduct of benzo[a]pyrene diol epoxide (BPDE), were synthesized and used to study the binding stoichiometry between the DNA adduct and its antibody. The free oligonucleotide and its complexes with mouse monoclonal antibody were separated using capillary electrophoresis and detected with laser-induced fluorescence (LIF). Two complexes, representing the 1:1 and 1:2 stoichiometry between the antibody and the DNA adduct, were clearly demonstrated.
Analysis of DNA-ligand binding in solution and on biochips
Biophysics, 2011
Ligand binding to DNA, as well as to microarrays, requires a system approach to description and analysis. This type of approach implies a fixed sequence of operations. Firstly, it is necessary to make a description of a binding scheme that realizes ligand and polymer in common spatial way. Secondly, a physical model of binding is required. Thirdly, a mathematical binding model should be constructed on the basis of the binding scheme and the physical model of binding. Every analysis of experimental data needs this prelim inary work. A mathematical apparatus and classification of binding models have to follow on. Classification of different binding isotherms by different binding models is the direct problem. The inverse problem is a reconstruction of parameters of a binding model by experimental binding isotherm curves. The inverse prob lem can only be solved after solving the direct problem. An example of classification of binding models by oli gonucleotides or proteins binding cooperativity and polymer properties like homo or heteropolymer is pre sented.
The Journal of Physical Chemistry B, 2010
The mode of binding of 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) with calf thymus DNA as revealed from different steady state and time-resolved emission spectroscopic measurements has been reported in this paper. Fluorescence enhancement of DASPMI and its quenching by potassium iodide (KI) points to groove binding of dye with ct-DNA, rather than intercalation in the ct-DNA helix. An increase in steady state anisotropy and fluorescence lifetime hints at binding with ct-DNA. The value of binding constant from emission and association constant from circular dichroic spectrum also indicates weak binding. The strong dependence on ionic strength or salt in controlling the binding of DASPMI with ct-DNA by electrostatic interaction confirms groove binding. The high semicone angle of DASPMI in ct-DNA certainly rules out the possibility of intercalated bonding. A theoretical modeling shows that the probe is bound to ct-DNA as a crescent with a curvature of 11.35 Å, which is the previously known curvature of probe in the minor groove.
Journal of Drug Metabolism & Toxicology, 2016
The double helical structure of DNA offers various binding sites for the interaction of ligands or proteins. Interactions using minor groove, major groove, and through intercalation are the major types of binding mechanisms of DNA-ligand interactions. The lowering in the absorption intensity along with bathochromic shift is the indication of intercalation binding mode of the dye into the base pairs of the DNA. In this study, the interaction of phenothiazine dyes with calf-thymus DNA (ctDNA) in physiological buffer (pH 7.4) was studied using UV-visible, fluorescence, circular dichroism (CD), and UV-thermal denaturation spectroscopy. The binding constants were calculated at different temperatures with the help of fluorescence spectroscopy. CD signals signify that B-form of DNA might become more compact, upon binding of the dyes. Also, induced circular dichroism is observed which confirms the dye-DNA complex formation. Stabilization of DNA double helix upon binding with dyes was confirmed by the increase in T m of ctDNA. Based on thermal melting profiles, it was found that thionine acetate is most promising in stabilizing the DNA double helix, in comparison to other two dyes. Also, binding constants calculated by fluorescence is in accordance with the thermal melting analysis. These results are indicative of the intercalation binding mode between dyes and the DNA. The binding affinity of the dyes to DNA is found to be in order as thionine acetate > azure A > azure B. Such preliminary studies facilitate our understanding about various types of DNAligand interactions and provide clues for designing new and more effective drugs.
Binding Expedient of 2-carbamido-1,3-indandione to Nucleic Acids: Potential Fluorescent Probe
Photochemistry and Photobiology , 2021
Fluorescent and computational methods were used to elucidate the binding expedient of 2-carbamido-1,3-indandione (CAID) tautomers to nucleotides. The dependence of the fluorescence emission of CAID loaded nucleic acids sequences to compound concentration, temperature and time variation was investigated. It was found that the subject compound binds to nucleic acids but does not intercalate. According to our quantum-chemical calculations on the conjugation between CAID and nucleotides, the binding in the formed complexes may be through hydrogen bonds. Two possible types of complexes were considered-CAID to the phosphate group and CAID to the nucleobase. To estimate the binding affinity, the interaction energies of the formed complexes were calculated. Tautomer 2-carboamide-1-hydroxy-3-oxo-indane is preferred in the formation of complexes, and the phosphate group complexes were more stable. Generally, the guanosine and deoxyguanosine monophosphate complexes were the most preferred regardless of the complex type. Because of the lack of cytotoxic effect on untransformed cell lines of mouse embryo fibroblasts Balb/c 3T3 according to our previous report (Markova et al, (2017) Bulg Chem Commun, 49D, 221-226) and the affinity to nucleic acids, we can suggest that the subject compound could be suitable to be used as a novel type of fluorescent biomarker.
Environment-induced changes in DNA conformation as probed by ethidium bromide fluorescence
Biophysical Chemistry, 1990
The interaction of the ethidium cation with calf thymus DNA is investigated in solutions of different ionic strength and temperature by observation of the enhancement of fluorescence of ethidium upon intercalation in the duplex structure. The quantum yield of the fluorescence of the intercalated dye is found to increase either upon lowering the NaC concentration or upon increasing the temperature. The existence of a correlation between the geometry of the intercalation complex and the features of the secondary structure of DNA is suggested. Binding isotherms under corresponding environmental conditions are also quantitated by fluorescence enhancement and interpreted in terms of the neighbor exclusion model. Large contributions from change in hydration to the thermodynamics of binding are demonstrated by the temperature dependences of the equilibrium constants. The neighbor exclusion range is found to be practically independent of the salt concentration but its value increases from an average of 2.4 around room temperature to 4-5 at 80°C, as inferred from the binding curves in 0.15 and 0.5 M [Na+] or from the DNA hypochromism vs temperature profiles of complexes at 10-3 M [Na* 1. All the data point to a possible sequence-conformation specificity in the intercalation of ethidium which in heterogeneous DNA is mediated by environmental changes.
Photochemistry and Photobiology, 2001
The absorption and fluorescence spectra, fluorescence quantum yields, lifetimes and time-resolved fluorescence spectra are reported for nine different fluorescent DNAdyes. The work was initiated in search of a quantitative method to detect the ratio of single-to-double stranded DNA (ssDNA/dsDNA) in solution based on the photophysics of dye-DNA complexes; the result is a comprehensive study providing a vast amount of information for users of DNA stains. The dyes examined were the bisbenzimide or indole-derived stains (Hoechst 33342, Hoechst 33258 and 4,6-diamidino-2-phenylindole), phenanthridinium stains (ethidium bromide and propidium iodide) and cyanine dyes (PicoGreen, YOYO-1 iodide, SYBR Green I and SYBR Gold). All were evaluated under the same experimental conditions in terms of ionic strength, pH and dye-DNA ratio. Among the photophysical properties evaluated only fluorescence lifetimes for the cyanine stilbene dyes allowed a convenient differentiation between ssDNA and dsDNA. The bisbenzimide dyes showed multiexponential decays when bound to either form of DNA, making lifetime-based analysis cumbersome with inherent errors. These dyes also presented biexponential decay when free in aqueous buffered solutions at different pH. A mechanism for their deactivation is proposed based on two different conformers decaying with different kinetics. The phenanthridinium dyes showed monoexponential decays with ssDNA and dsDNA, but there was no discrimination between them. High dye-DNA ratios (e.g. 1:1) resulted in multiexponential decays for cyanine dyes, resulting from energy transfer or self-quenching deactivation. Shifts in both absorption and fluorescence maxima for both ssDNA and ds-DNA DNA-cyanine dye complexes were small. Broadening of dye-ssDNA absorption and fluorescence bands for the cyanine dyes relative to dye-dsDNA bands was detected and attributed to higher degrees of rotational freedom in the former. ¶Posted on the website on 23 March 2001.