Interaction of actinomycin D, ethidium, quinacrine, daunorubicin, and tetralysine with DNA: 31P NMR chemical shift and relaxation investigation.

The binding of actinomycin D, ethidium, quinacrine, daunorubicin, and tetralysine to DNA has been investigated using 31P NMR. Titration of DNA with actinomycin yields a new downfield peak or overlapping peaks as would be expected from the slow dissociation kinetics of this compound. The other intercalators shift the DNA 31P signal downfield as a single exchange averaged peak. Tetralysine causes a slight upfield shift. The chemical shift titration curves for the intercalators are sigmoid curves suggesting that cooperative processes or competing effects on the chemical shift are being observed. The magnitude of the chemical shift change at saturation of DNA with the compounds is found to vary significantly and to be linearly related to the DNA base pair unwinding angle for the compounds. Analysis of 31P spin lattice relaxation times (T1) and linewidths as a function of temperature (below Tm) and titration with the above compounds indicates that T1 does not change significantly while linewidth increases with decreasing temperature and increasing bound intercalator. One interpretation of these results is that in both cases the overall motion of DNA becomes slower while the internal motion is not greatly affected.     

Comparative viscometric analysis of the interaction of chloroquine and quinacrine with superhelical and sonicated DNA.

Unwinding angles for the structurally related antimalarial drugs chloroquine and quinacrine have been determined with superhelical Col E1 plasmid DNA by applying the quantitative method developed by Vinograd and co-workers (Revet, B.M., Schmir, M. and Vinograd, J. (1971) Nat. New Biol. 229, 10). The value for chloroquine, 8.6 degrees, calculated assuming an unwinding angle of 26 degrees for ethidium bromide, is significantly lower than the value for quinacrine, 22.5 degrees, calculated in the same manner. Viscometric titrations with sonicated calf thymus DNA were quantitated using available binding constants for the two drugs and indicated that chloroquine also causes significantly smaller DNA length increases on intercalation relative to quinacrine. The conclusion from these experiments is that chloroquine does not bind to DNA by the classical intercalation mechanism typical of quinacrine and ethidium.     

Phosphorus-31 nuclear magnetic resonance of ethidium complexes with ribonucleic acid model systems and phenylalanine-accepting transfer ribonucleic acid

The temperature dependence of the 31P NMR spectra of the ethidium complexes with poly(A) X oligo(U) and the 31P spectra of phenylalanine tRNA (yeast) in various molar ratios of ethidium ion (Et) are presented. In the poly(A) X oligo(U) X Et complex, a new peak about 2.0 ppm downfield from the double-helix peak appears. We have assigned this peak to phosphates perturbed by ethidium. The chemical shift of this peak is consistent with the intercalation mode of binding and provides additional support for our hypothesis that 31P shifts are sensitive probes of phosphate ester conformations. The main effect of ethidium on the 31P spectra of tRNAPhe is the broadening of several of the scattered signals. These scattered signals are associated with phosphates involved in tertiary interactions. We propose that these broadened signals arise from phosphates near the Et binding site.     

Binding mode of 4',6-diamidino-2-phenylindole and ethidium to poly(dG).poly(dC).poly(dC)(+) triplex and poly(dG).poly(dC) duplex

Optical spectroscopic properties of 4',6-diamidino-2-phenylindole (DAPI) and ethidium bromide complexed with poly(dG).poly(dC).poly(dC)(+) triplex and poly(dG).poly(dC) duplex were compared in this study. When complexed with both duplex and triplex, ethidium is characterized by hypochromism and a red shift in the absorption spectrum, a complicate induced circular dichroism (CD) band in the polynucleotide absorption region, and a negative reduced linear dichroism signal in both polynucleotide and drug absorption regions. The spectral properties for both duplex- and triplex-bound ethidium are identical and both can be understood by the intercalation binding mode. In contrast, the absorption and CD spectra of DAPI complexed with triplex differ from those of the DAPI-duplex complex, although both complexes can be understood by the intercalation binding mode. Considering that the third strand runs along the major groove of the template duplex, we conclude that the DAPI molecule partially intercalates near the major groove of the duplex, where the third strand can affect its spectroscopic properties.     

Studies on amikhellin: I. Intercalative binding to double-stranded DNA

The present paper reports that amikhellin, a drug so far used as a coronary vasodilator, binds to double-stranded DNA by an intercalation process which does not depend upon DNA base composition. The binding to DNA was established by spectrophotometry, ultracentrifugation and competition with ethidium bromide. The parameters of the binding equilibrium were calculated by these two latter methods. Evidence for intercalation was obtained from the observation by viscosimetric experiments of the length increase of sonicated calf thymus DNA and of the untwisting of circular PM2 DNA. The unwinding angle was measured to be 6° per bound drug molecule.     

The binding of echinomycin to deoxyribonucleic acid.

Echinomycin is a peptide antibiotic which binds strongly to double-helical DNA up to a limit of approximately one molecule per five base-pairs. There is no detectable interaction with rRNA and only extremely feeble non-specific interaction with poly(rA)-poly(rU). Heat denaturation of DNA greatly decreases the binding, and similarly limited interaction is observed with naturally occurring single-stranded DNA. Association constants for binding to nine double-helical DNA species from different sources are presented; they vary by a factor of approximately 10, but are not simply related to the gross base composition. The interaction with DNA is ionic-strength-dependent, the binding constant falling by a factor of 4 when the ionic strength is raised from 0.01 to 0.10mol/litre. From the effect of temperature on the association constant for calf thymus DNA, the enthalpy of interaction is calculated to be about -13kJ/mol (-3kcal/mol). Binding of echinomycin persists in CsCl gradients and the buoyant density of nicked bacteriophage PM2 DNA is decreased by 25 mg/ml. Echinomycin interacts strongly with certain synthetic poly-deoxynucleotides, the binding constant decreasing in the order poly(dG)-poly(dC) greater than poly(dG-dC) greater than poly(dA-dT). For the latter two polymers the number of base-pairs occluded per bound antibiotic molecule is calculated to be three, whereas for poly(dG)-poly(dC) it is estimated to be four to five. Poly(dA)-poly(dT) and poly(dI)-poly(dC) interact only very weakly with the antibiotic. Poly(dI-dC) interacts to a slightly greater extent, but the binding curve is quite unlike that seen with the three strongly binding synthetic polynucleotides. Echinomycin affects the supercoiling of closed circular duplex bacteriophage PM2 DNA in the characteristic fashion of intercalating drugs. At low ionic strength the unwinding angle is almost twice that of ethidium. Likewise the extension of the helix, determined from changes in the viscosity of rod-like sonicated DNA fragments, is nearly double that expected for a simple (monofunctional) intercalation process. On this basis the interaction process is characterized as bifunctional intercalation. At higher ionic strength the unwinding angle relative to that of ethidium and the helix extension per bound echinomycin molecule fall, indicating a smooth progression towards more nearly monofunctional intercalation. Two simpler compounds which act as analogues of the quinoxaline chromophores of echinomycin, quinoxaline-2-carboxamide and the trypanocidal drug Bayer 7602, interact with DNA very much more weakly than does echinomycin, showing that the peptide portion of the antibiotic plays an essential role in determining the strength and specificity of the interaction.     

Imino 1H- and 31P-NMR analysis of the interaction of propidium and ethidium with DNA

At low temperature and low salt concentration, both imino proton and ³¹p-nmr spectra of DNA complexes with the intercalators ethidium and propidium are in the slow-exchange region. Increasing temperature and/or increasing salt concentration results in an increase in the site exchange rate. Ring-current effects from the intercalated phenanthridinium ring of ethidium and propidium cause upfield shifts of the imino protons of A · T and G · C base pairs, which are quite similar for the two intercalators. The limiting induced chemical shifts for propidium and ethidium at saturation of DNA binding sites are approximately 0.9 ppm for A · T and 1.1 ppm for G · C base pairs. The similarity of the shifts for ethidium and propidium, in both the slow- and fast-exchange regions over the entire titration of DNA, shows that a binding model for propidium with neighbor-exclusion binding and negative ligand cooperativity is correct. The fact that a unique chemical shift is obtained for imino protons at intercalated sites over the entire titration and that no unshifted imino proton peaks remain at saturation binding of ethidium and propidium supports a neighbor-exclusion binding model with intercalators bound at alternating sites rather than in clusters on the double helix. Addition of ethidium and propidium to DNA results in downfield shifts in ³¹P-nmr spectra. At saturation ratios of intercalator to DNA base pairs in the titration, a downfield shoulder (approximately ?2.7 ppm) is apparent, which accounts for approximately 15% of the spectral area. The main peak is at ?3.9 to ?4.0 ppm relative to ?4.35 in uncomplexed DNA. The simplest neighbor-binding model predicts a downfield peak with approximately 50% of the spectral area and an upfield peak, near the chemical shift for uncomplexed DNA, with 50% of the area. This is definitely not the case with these intercalators. The observed chemical shifts and areas for the DNA complexes can be explained by models, for example, that involve spreading the intercalation-induced unwinding of the double helix over several base pairs and/or a DNA sequence- and conformation-dependent heterogeneity in intercalation-induced chemical shifts and resulting exchange rates.     

Quinacrine: Spectroscopic properties and interactions with polynucleotides

The acridine dye quinacrine and its interactions with calf thymus DNA, poly(dA-dT) · poly (dA-dT), and poly (dG-dC) · poly(dG-dC) were studied by light absorption, linear dichroism, and fluorescence spectroscopy. The transition moments of quinacrine give rise to absorption bands polarized along the short axis (400–480-nm band), and the long axis (345-nm and 290-nm bands) of the molecule, respectively. Linear dichroism studies show that quinacrine intercalates into calf thymus DNA as well as into the polynucleotides, displaying fairly homogeneous binding to poly (dA-dT) · poly (dA-dT), but more than one type of intercalation site for calf thymus DNA and poly (dG-dC) · poly(dG-dC). Fluorescence spectroscopy shows that for free quinacrine the pK = 8.1 between the mono- and diprotonated states also remains unchanged in the excited state. Quinacrine bound to calf thymus DNA and polynucleotides exhibits light absorption typical for the intercalated diprotonated form. The fluorescence enhancement of quinacrine bound to poly (dA-dT) · poly(dA-dT) may be due to shielding from water interactions involving transient H-bond formation. The fluorescence quenching in poly(dG-dC) · poly(dG-dC) may be due to excited state electron transfer from guanine to quinacrine. © 1993 John Wiley & Sons, Inc.     

Modulation of chromatin superstructure induced by poly(ADP-ribose) synthesis and degradation

It has been demonstrated recently by Poirier et al. (Poirier, G. G., de Murcia, G., Jongstra-Bilen, J., Niedergang, C., and Mandel, P. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3423-3427) that poly(ADP-ribosyl)ation of pancreatic nucleosomes causes relaxation of the chromatin superstructure through H1 modification. The in vitro effect of poly(ADP-ribose) synthesis and degradation on calf thymus chromatin was investigated by the time course incorporation of ADP-ribose, electron microscopy, analytical ultracentrifugation, and autoradiography of the protein acceptors. Purified calf thymus poly(ADP-ribose) polymerase and partially purified bull testis poly(ADP-ribose) glycohydrolase were used. Degradation of ADP-ribose units on hyper(ADP-ribosyl)ated H1 by poly(ADP-ribose) glycohydrolase restores the native condensed chromatin superstructure. This reversible conformational change induced by poly(ADP-ribosyl)ation on nucleosomal arrangement could be one of the mechanisms by which the accessibility of DNA polymerases and/or excision-repair enzymes is favored, the native structure being fully restorable.     

Induced circular dichroism in nucleic acid-acridine derivative complexes

Visible absorption and circular dichroism (CD) spectra have been measured for complexes formed between nucleic acids (calf thymus DNA, poly(rA).poly(rU) and poly(rI).poly(rC)) and 9-aminoacridines (quinacrine, acranil and 9-amino-6-chloro-2-methoxy acridine). With poly(rA).poly(rU), a new absorption band was observed at longer wavelengths. The nucleic acid-drug complexes showed considerable different induced CD spectra. Analysis of these CD spectra suggests that the cationic side chains of quinacrine and acranil play an important role on the binding properties to DNA and poly(rA).poly(rU).     

Intercalative binding to DNA of antitumour drugs derived from 3-nitro-1,8-naphthalic acid.

Two new antitumour drugs, imide derivatives of 3-nitro-1,8-naphthalic acid having different basic side chains linked to the imide nitrogen, have been shown to bind to double-helical DNA by intercalation. At ionic strength 0.01 mol/litre, pH 7, their intrinsic association constants are about 1.45 x 10(5) M-1 and each bound ligand molecule occludes about 3.4 nucleotides of the DNA lattice. They remove and reverse the supercoiling of closed circular duplex PM2 DNA with apparent unwinding angles of 11-12 degrees per bound drug molecule, referred to an assumed unwinding angle of 26 degrees for ethidium. They increase the viscosity of sonicated rod-like DNA fragments, each bound drug molecule producing a calculated increment in length of 2.2 - 2.5 A. No important differences between the DNA-binding characteristics of the two drugs were detected, though one appears marginally more active than the other in certain biological tests.     

Protective action of copper (II) complex of a Schiff base against DNA damage induced by m-chloroperbenzoic acid using a novel DNA unwinding technique

DNA strand breaks can be detected with great sensitivity by exposing calf thymus DNA to alkaline solutions and monitoring the rate of strand unwinding. Fluorometric analysis of DNA unwinding (FADU) is a reliable method for detecting single-strand DNA breaks as an index of DNA damage induced by photosensitizer.m-Chloroperbenzoic acid (CPBA) was used as a photosensitizer in the photodamage of calf thymus DNA. When DNA is exposed to ionizing radiation, the radicals produced in the irradiated sample modify the base-pair regions of the double strands. The protective action of copper salt, Schiff base [ethylene diamine with ethyl acetate](L) and its Cu(II) complex (Cu(7) L Cl(14)) against DNA damage photoinduced by CPBA was studied using ethidium bromide as a fluorescent probe. Treatment of DNA with 5, 10, 50, 100, or 200 microM CPBA produced 75%, 48%, 38%, 32% and 30% double-stranded DNA remaining, respectively after 30 min of alkaline treatment at 15 degrees C. Treatment of calf thymus DNA irradiated with CPBA with a dose of 1 mM [Cu(7) L Cl(14)] produced 96% double-stranded remaining protection under the same conditions compared with irradiated DNA without addition of Cu(II) complex of Schiff base.     

Cooperative binding of a platinum metallointercalation reagent to poly(A).poly(U).

The cationic complex (2-hydroxyethanethiolato)(2,2',2'-terpyridine)platinum(II), [(terpy)Pt(HET)]+, binds cooperatively to poly(A).poly(U) by intercalation. The melting temperature of poly(A).poly(U) in low-salt buffer is increased by 6 degrees C in the presence of [(terpy)Pt(HET)]+, indicating stabilization of the duplex structure by the bound platinum reagent. Viscosity measurements provide evidence for comparable lengthening of the polynucleotide in the presence of [(terpy)Pt(HET)]+ and the intercalating dye, ethidium bromide. Scatchard plots of the binding of [(terpy)Pt(HET)]+ to poly(A).poly(U) and poly(I).poly(C), determined through ultracentrifugation pelleting methods, show large positive curvature, reflecting the strong cooperativity associated with the platinum complex-RNA interaction. The characteristics of the binding isotherms are interpreted in terms of a model where cooperative pair units of [(terpy)Pt(HET)]+ intercalate into the double-stranded polymer. At saturation, two platinum molecules are bound for every three base pairs. This stoichiometry may be compared with the nearest-neighbor-exclusion binding observed previously in the interaction of [(terpy)Pt(HET)]+ and the ethidium cation with DNA, in which one intercalator occupies every other interbase-pair site at saturation. The striking differences observed in the interaction of [(terpy)Pt(HET)]+ with DNA and RNA suggest that drug recognition is sensitive to the constraints imposed by nucleic acid secondary structure.     

The use of fluorescence anisotropy decay of poly d(A-T) ethidium bromide complex to estimate the unwinding angle of the double helix.

We measured the fluorescence decay under polarized light, of ethidium bromide bound to the poly d(A-T) isolated from Cancer Pagurus. The decay of the whole fluorescence is a single exponential function revealing a good homogeneity of the binding sites. The anisotropy decay due to energy transfers between the ethidium bromide molecules bound to a same poly d(A-T) molecule has been analysed, with a Monte Carlo calculation. We found the dye unwinds the poly d(A-T) duplex by an angle of 17 degrees plus or minus 2 degrees. This result is in agreement with the value previously found in the case of calf thymus DNA-ethidium bromide complex, although the base compositions of the two nucleic acids are different.     

DNA-drug interactions. The crystal structures of d(TGTACA) and d(TGATCA) complexed with daunomycin

The anticancer drug daunomycin has been co-crystallized with the hexanucleotide duplex sequences d(TGTACA) and d(TGATCA) and single crystal X-ray diffraction studies of these two complexes have been carried out. Structure solution of the d(TGTACA) and d(TGATCA) complexes to 1.6 and 1.7 Angstrom resolution, respectively, shows two daunomycin molecules bound to the DNA hexamer. Binding occurs via intercalation of the drug chromophore at the d(TpG) step, and hydrogen bonding interactions involving the drug, DNA and solvent molecules. The daunomycin sugar is located in the minor groove of the DNA hexamer and is stabilized by hydrogen bonds between the amino group of the sugar and functional groups on the floor of the groove. The amino sugar of the d(TGATCA) duplex interacts directly with the DNA sequence, while in the d(TGTACA) duplex, the interaction is via solvent molecules. Two other complexes d(CGTACG)-daunomycin and d(CGATCG)-daunomycin have previously been structurally characterized. Comparison of the four structures with daunomycin bound to the triplet sequences 5'TGT, 5'TGA, 5'CGT and 5'CGA reveals changes in the conformation of both the DNA hexamer and the daunomycin upon complexation, as well as the hydrogen bonding and van der Waals' interactions.     

Microcalorimetric investigation of DNA, poly(dA)poly(dT) and poly[d(A-C)]poly[d(G-T)] melting in the presence of water soluble (meso tetra (4 N oxyethylpyridyl) porphyrin) and its Zn complex

Thermodynamic parameters of melting process (DeltaHm, Tm, DeltaTm) of calf thymus DNA, poly(dA)poly(dT) and poly(d(A-C)).poly(d(G-T)) were determined in the presence of various concentrations of TOEPyP(4) and its Zn complex. The investigated porphyrins caused serious stabilization of calf thymus DNA and poly poly(dA)poly(dT), but not poly(d(A-C))poly(d(G-T)). It was shown that TOEpyp(4) revealed GC specificity, it increased Tm of satellite fraction by 24 degrees C, but ZnTOEpyp(4), on the contrary, predominantly bound with AT-rich sites and increased DNA main stage Tm by 18 degrees C, and Tm of poly(dA)poly(dT) increased by 40 degrees C, in comparison with the same polymers without porphyrin. ZnTOEpyp(4) binds with DNA and poly(dA)poly(dT) in two modes--strong and weak ones. In the range of r from 0.005 to 0.08 both modes were fulfilled, and in the range of r from 0.165 to 0.25 only one mode--strong binding--took place. The weak binding is characterized with shifting of Tm by some grades, and for the strong binding Tm shifts by approximately 30-40 degrees C. Invariability of DeltaHm of DNA and poly(dA)poly(dT), and sharp increase of Tm in the range of r from 0.08 to 0.25 for thymus DNA and 0.01-0.2 for poly(dA)poly(dT) we interpret as entropic character of these complexes melting. It was suggested that this entropic character of melting is connected with forcing out of H2O molecules from AT sites by ZnTOEpyp(4) and with formation of outside stacking at the sites of binding. Four-fold decrease of calf thymus DNA melting range width DeltaTm caused by increase of added ZnTOEpyp(4) concentration is explained by rapprochement of AT and GC pairs thermal stability, and it is in agreement with a well-known dependence, according to which DeltaT approximately TGC-TAT for DNA obtained from higher organisms (L. V. Berestetskaya, M. D. Frank-Kamenetskii, and Yu. S. Lazurkin. Biopolymers 13, 193-205 (1974)). Poly (d(A-C))poly(d(G-T)) in the presence of ZnTOEpyp(4) gives only one mode of weak binding. The conclusion is that binding of ZnTOEpyp(4) with DNA depends on its nucleotide sequence.     

Spectrophotometric and NMR analysis of the interaction of fluorinated mono- and bifunctional intercalators with DNA, poly(dA-dT), and poly(dG-dC)

We have synthesized and investigated the DNA binding properties of three fluorinated acridine derivatives—a monomer (I), a short dimer (II) and a long dimer (III). Only III has a sufficiently long chain bridging the two acridine nuclei to permit binding by bisintercalation. Analysis of the equilibrium and kinetic binding properties of these compounds to poly(dA-dT) demonstrates that they behave very similarly to their unfluorinated parent compounds. Helix extension, as determined by viscosity measurements, shows that both compounds I and II bind by monointercalation while III binds by bisintercalation. These results are confirmed by ¹⁹F-nmr analysis, which indicates, in particular, that the two chromophores of III share the same molecular environment as that of I in the presence of either calf thymus DNA or poly(dA-dT). Negative nuclear Overhauser effects in the presence of DNA indicate tight binding such that the motion of the ligands is governed by the polynucleotide dynamics. Optical titrations establish that in 4M NaCl, both I and III bind to calf thymus DNA, but no binding was observed with poly(dG-dC). This result is in contrast to those for dimers of ethidium, which show substantial binding to polynucleotides under high salt conditions. Nuclear magnetic resonance experiments, however, carried out at considerably higher concentrations, show that compound I does indeed bind to poly(dG-dC) under these high salt conditions, albeit weakly, and leads to a conversion of the polynucleotide from a left-handed to a right-handed conformation.     

Biphasic helix–coil transition of the ethidium bromide·poly(dA-dT) and the propidium diiodide·poly(dA-dT) Complexes. Stabilization of base-pair regions centered about the intercalation site

The nonexchangeable base and sugar proton nmr resonances and the 260 and 278-nm uv-absorbance bands of the nucleic acid were utilized to monitor the temperature-dependent duplex-to-strand transition of the alternating purine–pyrimidine deoxyribopolynucleotide poly(dA-dT) in the absence and presence of ethidium bromide (EB) at phosphate/drug = 50, 28, and 15 and propidium diiodide (PI) at P/D = 50, 25, 15, 10, and 5 in 0.1 M salt between 50° and 100°C. The nmr and optical methods monitor a biphasic duplex-to strand transition for the drug–poly(dA-dT) complexes. We have monitored the dissociation of the drug from the complex at the ethidium bromide phenanthridine ring and side-chain proton nmr resonances and the propidium diiodide 494 and 535-nm uv-absorbance bands and demonstrate that dissociation of the drug corresponds to the higher temperature transition in the biphasic nucleic acid melting curves. The lower temperature cooperative transition is assigned to the opening of drug-free AT base-pair regions in the drug–poly(dA-dT) complex and exhibits an increase in transition midpoint and a decrease in cooperativity with increasing drug concentration. The higher temperature cooperative transition is assigned to the opening of AT base-pair regions centered about the bound drug in the complex and exhibits an increase in the transition midpoint on raising the drug concentration. The large upfield shifts of the phenanthridine ring (but not side chain) protons of ethidium bromide on complex formation demonstrate intercalation of the drug between base pairs of the poly(dA-dT) duplex. The nucleic acid base and sugar resonances of poly(dA-dT) in 0.1 M phosphate undergo chemical shift changes between 0° and 50°C indicative of premelting conformational transition(s).     

Altered physiochemical properties of the deoxyribonucleic acid-mitomycin C complex. Evidence for the conformational change in deoxyribonucleic acid

Binding of the antibiotic mitomycin C to sonicated calf thymus DNA results in increased viscosity and an unaltered sedimentation constant of DNA. Flow dichroism measurements of the mitomycin C-DNA complex indicate that the 310-nm absorbance vector of the chromophore of the bound drug is oriented at approximately 57.2 degrees relative to the helix axis. A conclusion drawn from these results is that mitomycin C does not intercalate between base pairs, but rather, it is bound in one of the grooves. Binding of mitomycin C causes a number of changes which are DNA size dependent: (1) increased viscosity of sonicated, decreased viscosity of nonsonicate DNA; (2) unaltered sedimentation rate of sonicated, increased rate of nonsonicated DNA; (3) reduced electrophoretic mobility of nonsonicated DNA; (4) electron microscopic appearance of sonicated DNA-mitomycin complexes which is similar to that of control, while nonsonicated DNA complexes which display highly coiled, looped structures not seen in control nonsonicated DNA. These size-dependent effects are interpreted as indicative of conformational distortion of DNA at rare intervals, caused by a minor fraction of total bound mitomycin. The parallel used of sonicated and nonsonicated DNA as probes for certain effects of drug binding may be useful for detecting this type of phenomenon in general.