Ethidium bromide interactions with DNA: an exploration of a classic DNA–ligand complex with unbiased molecular dynamics simulations

Visualization of double stranded DNA in gels with the binding of the fluorescent dye ethidium bromide has been a basic experimental technique in any molecular biology laboratory for >40 years. The interaction between ethidium and double stranded DNA has been observed to be an intercalation between base pairs with strong experimental evidence. This presents a unique opportunity for computational chemistry and biomolecular simulation techniques to benchmark and assess their models in order to see if the theory can reproduce experiments and ultimately provide new insights. We present molecular dynamics simulations of the interaction of ethidium with two different double stranded DNA models. The first model system is the classic sequence d(CGCGAATTCGCG)₂ also known as the Drew–Dickerson dodecamer. We found that the ethidium ligand binds mainly stacked on, or intercalated between, the terminal base pairs of the DNA with little to no interaction with the inner base pairs. As the intercalation at the terminal CpG steps is relatively rapid, the resultant DNA unwinding, rigidification, and increased stability of the internal base pair steps inhibits further intercalation. In order to reduce these interactions and to provide a larger groove space, a second 18-mer DNA duplex system with the sequence d(GCATGAACGAACGAACGC) was tested. We computed molecular dynamics simulations for 20 independent replicas with this sequence, each with ∼27 μs of sampling time. Results show several spontaneous intercalation and base-pair eversion events that are consistent with experimental observations. The present work suggests that extended MD simulations with modern DNA force fields and optimized simulation codes are allowing the ability to reproduce unbiased intercalation events that we were not able to previously reach due to limits in computing power and the lack of extensively tested force fields and analysis tools.     

Selectively 13C-enriched DNA: Dynamics of the C1′-H1′ vector in d(CGCAAATTTGCG)2

Summary In order to examine the internal dynamic processes of the dodecamer d(CGCAAATTTGCG)₂, the ¹³C-enriched oligonucleotide has been synthesized. The three central thymines were selectively ¹³C-labeled at the C1′ position and their spin-lattice relaxation parameters R(C_Z), R(C_X,Y), R(H_Z→C_Z), R(2H_ZC_Z), R(2H_ZC_X,Y) and R(H _infZ ^supC ) were measured. Density functions were computed for two models of internal motions. Comparisons of the experimental data were made with the spin-lattice relaxation rates rather than with the density functions, whose values were altered by accumulation of the uncertainties of each relaxation rate measurement. The spin-lattice relaxation rates were computed with respect to the motions of the sugar around the C1′-N1 bond. A two-state jump model between the anti- and syn-conformations with P(anti)/P(syn)=91/9 or a restricted rotation model with Δχ=28° and an internal diffusion coefficient of 30×10⁷ s^-1 gave a good fit with the experimental data. Twist, tilt or roll base motions have little effect on ¹³C1′ NMR relaxation. Simulation of spin-relaxation rates with the data obtained at several temperatures between 7 and 32 °C, where the dodecamer is double stranded, shows that the internal motion amplitude is independent of the temperature within this range, as expected for internal motion. Using the strong correlation which exists in a B-DNA structure between the χ and δ angle, we suggest that the change in the glycosidic angle value should be indicative of a sugar puckering between the C1′-exo and C2′-endo conformations.     

Left-Handed Intercalated DNA Double Helix: Rendezvous of Ethidium and Actinomycin D in the Z-Helical Conformation Space

Abstract

It is now very well recognized that the DNA double helix is conformationally pluralistic and that this flexibility is derived from internal motions due to backbone torsions. But what is less apparent is that such internal motions can occur in a correlated fashion and express themselves in a wide variety of structural motifs and phenomena. For example, flexibility inherent in the DNA molecule can lead to a family of Z-DNA, LZ1 and LZ2 being the two extremes and correlated internal motion can cause LZ1?LZ2 transition. More interestingly, such motions manifest themselves as breathing modes on the DNA lattice resulting in the sequence specific intercalation sites. Following a detailed stereochemical analyses we observed that the intercalation site for ethidium is located at the dCpdG sequence of the intercalated LZ1 helix (LZ1*) while that for actinomycin D is located at the dGpdC sequence of the intercalated LZ2 helix (LZ2*). From the stereochemistry of the drug binding we make experimentally testable predictions which are in fact supported by a few recent experimental studies. These studies also show that a left-handed intercalated B-DNA model is a viable intermediate in the Z to B transition which can hold the drug with binding energy comparable to that of the intercalated right-handed B-DNA.     

Conformations and dynamics of surfactants in micelles and liquid crystals by nuclear magnetic relaxation

The order parameters as well as the rates of overall and internal motions of aggregated surfactants can be obtained from deuteron and carbon-13 nuclear relaxation experiments. The main contribution to the relaxation is generally the quadrupolar coupling (²H) or the short range dipolar interaction with protons (¹³C). In some cases it is convenient to derive the same information from the¹³C relaxation induced by long range dipolar interactions with a paramagnetic probe exchanging rapidly among the polar heads of surfactant molecules. This paper outlines the methods of interpretation of relaxation data by means of a rotational jump model of internal motions, taking into account most of the accessible conformers. The conformational and dynamical parameters are obtained from the magnetic field dependence of the longitudinal relaxation rates (micelles) or from the simultaneous fit of these rates and of the dipolar or quadrupolar splittings (liquid crystals). Some examples of application of these methods are given from recent works on single and double detailed surfactants.     

Internal DNA Modes Below 25 cm−1: A Resonance Raman Spectroscopy Observation

Abstract

The first resonance Raman scattering observation of the low-frequency (LF) region (below 40 up to 12 cm^?1) of DNA motions is presented. Since the concentration of the studied DNA solution was very low (1 mg/ml), the spectra features reflect internal vibrations of the macro-molecule. The decomposition of the spectra into Lorentzians clearly indicate three intrahelical DNA modes: the corresponding peaks are located at the frequencies 16,19, and 23 (±1) cm^?1. This result is in agreement with our quasi-continuity model of the LF B-form DNA dynamics (V. Lisy, P. Miskovsky and P. Schreiber, J. Biomol. Struct. Dyn. 13, 707 (1996)). The fit of the experimental frequencies to the theory, using the Genetic Algorithms approach, allowed us to make some conclusions about the model force constants which could be found by independent conformational energy calculations. Possible positions of five lowest-frequency DNA peaks, predicted by the model, are discussed.     

DNA Models for A,B, C and D Conformations Related to Fiber X-ray,Infrared and NMR Measurements

Abstract

A conformational analysis of the A, B, C and D DNA forms was made in order to establish molecular models presenting a good agreement with experimental data obtained from fiber X-ray, infrared linear dichroism and ³¹P NMR. The proposed models have been refined and do present good stereochemistry and optimized H-bond distances between bases associated with the Watson-Crick pairing. The DNA conformations proposed are a left handed double helix for the C form and right handed helices for A, B and D. Relations to conformational transitions between these forms are discussed.     

A critical evaluation of models for complex molecular dynamics: application of NMR studies of double- and single-stranded DNA

The nature of internal and overall motions in native (double-stranded) and denatured (single-stranded) DNA fragments 120–160 base pairs (bp) long is examined by molecular-dynamics modeling using ¹³C-nmr spin-relaxation data obtained over the frequency range of 37–125 MHz. The broad range of ¹³C frequencies is required to differentiate among various models. Relatively narrow linewidths, large nuclear Overhauser enhancements (NOEs), and short T₁ values all vary significantly with frequency and indicate the presence of rapid, restricted internal motions on the nanosecond time scale. For double-stranded DNA monomer fragments (147 bp, 24 Å diam at 32°C), the overall motion is that of an axially symmetric cylinder (τ_x = ～10^?6 s;τ_Z = ～1.8 × 10^?8s), which is in good agreement with values calculated from hydrodynamic theory (τ_x = ～1.8 × 10^?6 s; τ_Z = ～2.7 × 10^?8 s). The DNA internal motion can be modeled as restricted amplitude internal diffusion of individual C? H vectors of deoxyribose methine carbons C1′, C3′, and C4′, either with conic boundary conditions (τ_w = ～4 × 10^?9 s, θ_cone = ～21°) or as a bistable jump (τ_A = τ_B = ～2 × 10^?9 s, θ = ～15°). We discuss the critical role in molecular-dynamics modeling played by the angle (β) that individual C? H vectors make with the long axis of the DNA helix. Heat denaturation brings about increases in both the rate and amplitude of the internal motion (described by the wobble model with τ_W = ～0.2 × 10^?9 s, θ_cone = ～50°), and overall motion is affected by becoming essentially isotropic (τ_x = τ_Z = ～5 × 10^?8 s) for the single-stranded molecules. Since ¹³C-nmr data obtained at various DNA concentrations for C2′ of the deoxyribose ring is not described well by the above models, a new model incorporating an additional internal motion is proposed to take into account the rapid, extensive, and weakly coupled motion of C2′.     

Mode‐coupling Smoluchowski dynamics of a double‐stranded DNA oligomer

The local dynamics of a double‐stranded DNA d(TpCpGpCpG)₂ is obtained to second order in the mode‐coupling expansion of the Smoluchowski diffusion theory. The time correlation functions of bond variables are derived and the ¹³C‐nmr spin–lattice relaxation times T₁ of different ¹³C along the chains are calculated and compared to experimental data from the literature at three frequencies. The DNA is considered as a fluctuating three‐dimensional structure undergoing rotational diffusion. The fluctuations are evaluated using molecular dynamics simulations, with the ensemble averages approximated by time averages along a trajectory of length 1 ns. Any technique for sampling the configurational space can be used as an alternative. For a fluctuating three‐dimensional (3D) structure using the three first‐order vector modes of lower rates, higher order basis sets of second‐rank tensor are built to give the required mode coupling dynamics. Second‐ and even first‐order theories are found to be in close agreement with the experimental results, especially at high frequency, where the differences in T₁ for ¹³C in the base pairs, sugar, and backbone are well described. These atomistic calculations are of general application for studying, on a molecular basis, the local dynamics of fluctuating 3D structures such as double‐helix DNA fragments, proteins, and protein–DNA complexes. © 1999 John Wiley & Sons, Inc. Biopoly 50: 613–629, 1999     

A natural source of fluorescence depolarization in dye-DNA complexes

The interaction of ethidium bromide with intraphage (T₄) DNA and isolated phage (T₄) DNA has been studied. The partial polarizations of fluorescence of the dye-complexes have been measured at thermal equilibrium at various temperatures and by fast cooling to a constant lower temperature. A close comparison of the results at these two conditions and an additional analysis of them from Perrin's theorem prove that a natural source of depolarization is exhibitant in DNA-dye complexes at ordinary temperatures. This depolarization effect may be attributed to some internal motions or rotations in DNA. Alternatively, the effect may be due to conformational changes within the framework of the DNA double helix, which provide a different environment for the dye. The above depolarization effect is most effective in the temperature range 37–64°.     

Structure and dynamics of the complex of single stranded DNA binding protein of Escherichia coli with circular single stranded DNA of filamentous phages

We have analyzed the equilibrium and nonequilibrium properties of the complex of the single stranded DNA binding protein of Escherichia coli (EcoSSB) and circular single stranded DNA of filamentous phages M13mp8 and F1 using static and dynamic light scattering, analytical ultracentrifugation and electron microscopy. Upon binding to the single stranded DNA the EcoSSB tetramer replaces an equivalent volume of water trapped within the coiled single stranded DNA and hinders the folding of the single stranded DNA into secondary structures at all salt concentrations. The salt dependent compaction of the stoichiometric complex can be described assuming a flexible polyelectrolyte chain. The solution structure of the macromolecular complex is a random coil and in the electron microscope a beaded flexible structure of the complex with a bead diameter of 6 nm appears at all salt concentrations used. The internal motions of the stoichiometric complex can be described by the Rouse-Zimm model of polymer dynamics. The segmental mobility of the complex can be correlated with changes in the binding site size of the EcoSSB tetramer; it indicates the presence of interactions between EcoSSB tetramers bound to single stranded DNA.     

Local dynamics of DNA probed with optical absorption spectroscopy of bound ethidium bromide.

We have studied the local dynamics of calf thymus double-helical DNA by means of an "optical labeling" technique. The study has been performed by measuring the visible absorption band of the cationic dye ethidium bromide, both free in solution and bound to DNA, in the temperature interval 360-30 K and in two different solvent conditions. The temperature dependence of the absorption line shape has been analyzed within the framework of the vibronic coupling theory, to extract information on the dynamic properties of the system; comparison of the thermal behavior of the absorption band of free and DNA-bound ethidium bromide gave information on the local dynamics of the double helix in the proximity of the chromophore. For the dye free in solution, large spectral heterogeneity and coupling to a "bath" of low-frequency (soft) modes is observed; moreover, anharmonic motions become evident at suitably high temperatures. The average frequency of the soft modes and the amplitude of anharmonic motions depend upon solvent composition. For the DNA-bound dye, at low temperatures, heterogeneity is decreased, the average frequency of the soft modes is increased, and anharmonic motions are hindered. However, a new dynamic regime characterized by a large increase in anharmonic motions is observed at temperatures higher than approximately 280 K. The DNA double helix therefore appears to provide, at low temperatures, a rather rigid environment for the bound chromophore, in which conformational heterogeneity is reduced and low-frequency motions (both harmonic vibrations and anharmonic contributions) are hindered. The system becomes anharmonic at approximately 180 K; however, above approximately 280 K, anharmonicity starts to increase much more rapidly than for the dye free in solution; this can be attributed to the onset of wobbling of the dye in its intercalation site, which is likely connected with the onset of (functionally relevant) DNA motions, involving local opening/unwinding of the double helix. As shown by parallel measurements of the melting curves, these motions precede the melting of the double helix and depend upon solvent composition much more than does the melting itself.     

Effect of alkylated and intercalated DNA on the generation of superoxide anion by riboflavin

Superoxide anion (O ₂ ^.– ) was photogenerated upon illumination of riboflavin in fluorescent light. The rate of O ₂ ^.– formation was stimulated by double stranded DNA but not by denatured DNA or RNA. Depurinated DNA, which was predominantly depleted in guanine residues, did not exhibit the stimulatory effect, indicating an interaction of riboflavin, or active oxygen species derived from it, with guanine bases. Also, the stimulation of O ₂ ^.– photogeneration was not observed with ethidium bromide but was seen with proflavin-intercalated DNA. Since ethidium bromide intercalates preferentially between purines and pyrimidines, and proflavin prefers dA-dT rich sites, these results were interpreted to suggest that the interaction of riboflavin with DNA is mainly with GC or CG base pairs.     

Preparation, resonance assignment, and preliminary dynamics characterization of residue specific 13C/15N-labeled elongated DNA for the study of sequence-directed dynamics by NMR

DNA is a highly flexible molecule that undergoes functionally important structural transitions in response to external cellular stimuli. Atomic level spin relaxation NMR studies of DNA dynamics have been limited to short duplexes in which sensitivity to biologically relevant fluctuations occurring at nanosecond timescales is often inadequate. Here, we introduce a method for preparing residue-specific ¹³C/¹⁵N-labeled elongated DNA along with a strategy for establishing resonance assignments and apply the approach to probe fast inter-helical bending motions induced by an adenine tract. Preliminary results suggest the presence of elevated A-tract independent end-fraying internal motions occurring at nanosecond timescales, which evade detection in short DNA constructs and that penetrate deep (7 bp) within the DNA helix and gradually fade away towards the helix interior.     

Nucleic Acid Analog Peptide Containing β-Aminoalanine Modified with Nucleobases

Abstract

Oligopeptides containing N^β-(thymin-1-ylacetyl) β-aminoalanine and N^β-(cytosin-1-ylacetyl) β-aminoalanine moieties synthesized on solid phase using standard boc-chemistry showed hybridization properties with single stranded DNA and RNA, and also with double stranded DNA at pH 7.0.     

Hydration dependent dynamics in RNA

The essential role played by local and collective motions in RNA function has led to a growing interest in the characterization of RNA dynamics. Recent investigations have revealed that even relatively simple RNAs experience complex motions over multiple time scales covering the entire ms–ps motional range. In this work, we use deuterium solid-state NMR to systematically investigate motions in HIV-1 TAR RNA as a function of hydration. We probe dynamics at three uridine residues in different structural environments ranging from helical to completely unrestrained. We observe distinct and substantial changes in ²H solid-state relaxation times and lineshapes at each site as hydration levels increase. By comparing solid-state and solution state ¹³C relaxation measurements, we establish that ns–μs motions that may be indicative of collective dynamics suddenly arise in the RNA as hydration reaches a critical point coincident with the onset of bulk hydration. Beyond that point, we observe smaller changes in relaxation rates and lineshapes in these highly hydrated solid samples, compared to the dramatic activation of motion occurring at moderate hydration.     

Mapping the dynamics of ligand reorganization via 13CH3 and 13CH2 relaxation dispersion at natural abundance

Flexible ligands pose challenges to standard structure-activity studies since they frequently reorganize their conformations upon protein binding and catalysis. Here, we demonstrate the utility of side chain ¹³C relaxation dispersion measurements to identify and quantify the conformational dynamics that drive this reorganization. The dispersion measurements probe methylene ¹³CH₂ and methyl ¹³CH₃ groups; the latter are highly prevalent side chain moieties in known drugs. Combining these side chain studies with existing backbone dispersion studies enables a comprehensive investigation of μs–ms conformational dynamics related to binding and catalysis. We perform these measurements at natural ¹³C abundance, in congruence with common pharmaceutical research settings. We illustrate these methods through a study of the interaction of a phosphopeptide ligand with the peptidyl-prolyl isomerase, Pin1. The results illuminate the side-chain moieties that undergo conformational readjustments upon complex formation. In particular, we find evidence that multiple exchange processes influence the side chain dispersion profiles. Collectively, our studies illustrate how side-chain relaxation dispersion can shed light on ligand conformational transitions required for activity, and thereby suggest strategies for its optimization.     

Phosphorus NMR spectra of natural DNA fragments in the course of the B-to-A conformational transition

Changes in the ³¹P-nmr spectra of sonicated natural DNA fragments were investigated in ethanol solutions where the fragments underwent, as checked by CD, the B-to-A conformational transition. The study produced the following conclusions: (1) The high DNA concentrations used for the ³¹P-nmr measurements promote the transition compared to dilute solutions that are commonly used for CD measurements. (2) The B-to-A transition was reflected in a cooperative downfield shift of the DNA ³¹P-nmr resonance, consistent with unwinding of the double helix. (3) Prior to the transition, the changes in chemical shift of double-and single-stranded DNAs were almost identical. It thus appears that the effect of ethanol on the geometry and hydration of phosphodiester linkages does not depend heavily on DNA base–base interactions. (4) The A-form resonances were 30–40% narrower than the B-form resonances, which is attributed to marked sequence-dependent variations in the latter conformation and to their reduction in the former. (5) The B-form DNA aggregated in the concentrated ³¹P-nmr samples in the presence of ethanol, judged from a milky opalescence of the solution and a substantial broadening of its ³¹P-nmr resonance. The broadening abruptly disappeared as soon as DNA adopted the A-form so that DNA, in dependence on the secondary structure, showed different tendencies to condense in the presence of ethanol. The condensation increased cooperativity of the B-to-A interconversion.     

Dynamic Structure of Proteins in Solid State. 1H and 13C NMR Relaxation Study

Abstract

Temperature dependencies of ¹H non-selective NMR T₁ and T₂ relaxation times measured at two resonance frequencies and natural abundance ^l3C NMR relaxation times T_l and T_lr measured at room temperature have been studied in a set of dry and wet solid proteins—;Bacterial RNase, lysozyme and Bovine serum albumin (BSA). The proton and carbon data were interpreted in terms of a model supposing three kinds of internal motions in a protein. These are rotation of the methyl protons around the axis of symmetry of the methyl group, and fast and slow oscillations of all atoms. The correlation times of these motions in solid state are found around 10^?11, 10^?9 and 10^?6 s, respectively. All kinds of motion are characterized by the inhomogeneous distribution of the correlation times. The protein dehydration affects only the slow internal motion. The amplitude of the slow motion obtained from the carbon data is substantially less than that obtained from the proton data. This difference can be explained by taking into account different relative inter- and intra- chemical group contributions to the proton and carbon second moments. The comparison of the solid state and solution proton relaxation data showed that the internal protein dynamics in these states is different: the slow motion seems to be few orders of magnitude faster in solution.     

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.