Ethidium Bromide Intercalation Research Articles

The Intercalator Ethidium Bromide Generates Covalent Adducts at Apurinic/Apyrimidinic Sites in DNA.

Ethidium bromide was first described as a DNA intercalator 60 years ago and, over the ensuing years, may be the most widely used fluorescent DNA stain in molecular biology, biochemistry, and histology. Noncovalent DNA binding by ethidium has been well characterized, but to date, there have been no reports of covalent DNA adduct formation by ethidium bromide. This report describes the characterization of covalent adducts generated by the reaction of ethidium with apurinic/apyrimidinic (AP) sites in DNA. Adduct formation proceeds via the reaction of the amino group(s) on ethidium with the ring-opened aldehyde residue of the AP site in DNA to yield an imine. Ethidium-AP adducts may form under a variety of circumstances due to the ubiquitous occurrence of AP sites in cellular and synthetic DNA.

Predicting the effect of binding molecules on the shape and mechanical properties of structured DNA assemblies

Chemo-mechanical deformation of structured DNA assemblies driven by DNA-binding ligands has offered promising avenues for biological and therapeutic applications. However, it remains elusive how to effectively model and predict their effects on the deformation and mechanical properties of DNA structures. Here, we present a computational framework for simulating chemo-mechanical change of structured DNA assemblies. We particularly quantify the effects of ethidium bromide (EtBr) intercalation on the geometry and mechanical properties of DNA base-pairs through molecular dynamics simulations and integrated them into finite-element-based structural analysis to predict the shape and properties of DNA objects. The proposed model captures various structural changes induced by EtBr-binding such as shape variation, flexibility modulation, and supercoiling instability. It enables a rational design of structured DNA assemblies with tunable shapes and mechanical properties by binding molecules.

In-vitro and in-silico analysis and antitumor studies of novel Cu(II) and V(V) complexes of N-p-Tolylbenzohydroxamic acid

Copper(L2Cu) and vanadium(L2VOCl) complexes of N-p-tolylbenzohydroxamic acid (LH) ligand have been investigated for DNA binding efficacy by multiple analytical, spectral, and computational techniques. The results revealed that complexes as groove binders as evidenced by UV absorption. Fluorescence studies including displacement assay using classical intercalator ethidium bromide as fluorescent probe also confirmed as groove binders. The viscometric analysis too supports the inferences as strong groove binders for both the complexes. Molecular docking too exposed DNA as a target to the complexes which precisely binds L2Cu, in the minor groove region while L2VOCl in major groove region. Molecular dynamic simulation performed on L2Cu complex revealing the interaction of complex with DNA within 20 ns time. The complex stacked into the nitrogen bases of oligonucleotides and the bonding features were intrinsically preserved for longer simulation times. In-vitro cytotoxicity study was undertaken employing MTT assay against the breast cancer cell line (MCF-7). Potential cytotoxic activities were observed for L2Cu and L2VOCl complexes with IC50 values of showing 71 % and 74 % of inhibition respectively.

Thermodynamic and structural investigation of the interaction of quaternized 2,3-octakis-[(2-mercaptopyridine)phthalocyaninato] copper (II) sulfate (CuPc) with parallel and hybrid type G-quadruplex.

G-quadruplexes are important drug targets and get attention due to their existence in telomere, ribosomal DNA, promoter regions of some oncogenes, and the untranslated regions of mRNA. Due to the biological roles of G-quadruplexes, investigating of the G-quadruplex-small molecule interaction is essential. The primary motivation for these studies is the possibility of inhibiting cell functions associated with G-quadruplex sequences by binding with small molecules. Targeting the small molecules to desired tissue with the G-quadruplex vehicles is the second important goal of the G-quadruplex-small molecule interaction studies. In the present study, the new peripherally 2-mercaptopyridine octasubstituted copper(II) phthalocyanine and its quaternized derivative (CuPc) were synthesized and characterized by elemental analysis FT-IR, UV-Vis, and mass spectra. The excellent solubility of CuPc in water is essential for its transport in the organism. Because of this feature, its affinity toward G-quadruplex forming aptamers, AS1411, Tel21, and Tel45, was investigated. The UV-Vis spectrophotometric titration data confirmed the prevention of aggregation upon interaction with G-quadruplex, which is very important for biomedical applications. The CD spectroscopic analyses and binding stoichiometry confirmed the "end stacking" model for interaction of AS1411 with CuPc. The interaction of CuPc caused the equilibrium shift from hybrid conformation to antiparallel conformation for Tel21 and Tel45. The isothermal titration calorimeter (ITC) was used for the determination of thermodynamic parameters. The thermodynamic data of the interaction was fitted well with the one-site model. The negative values of Gibbs free energy change confirmed the spontaneous nature of the reactions. Besides, the negative values of enthalpy change and entropy change proved that the nature of processes was "enthalpy driven." The interaction stoichiometry was 2 for AS1411 and Tel21 and 1.5 for Tel45. The binding constants were 1.3(±0.3) × 105 , 3.2(±0.4) × 105 , and 1.1(±0.3) × 105 M-1 , which were at the level of ethidium bromide intercalation binding constant given in the literature. The DNA polymerase stop assay further supported the interaction of CuPc with G-quadruplex DNA. The experimental results confirm that the CuPc has a potential photosensitizer behaviour for photodynamic therapy.

Anti-toxoplasma activity and DNA-binding of copper(II) and zinc(II) coordination compounds with 5-nitroimidazole-based ligands.

Tetrahedral copper(II) and zinc(II) coordination compounds from 5-nitroimidazole derivatives, viz. 1-(2-chloroethyl)-2-methyl-5-nitroimidazole (cenz) and ornidazole 1-(3-chloro-2-hydroxypropyl)-2-methyl-5-nitroimidazole (onz), were synthesized and spectroscopically characterized. Their molecular structures were determined by X-ray diffraction studies. The complexes [Cu(onz)2X2], [Zn(onz)2X2], [Cu(cenz)2X2] and [Zn(cenz)2X2] (X- = Cl, Br), are stable in solution and exhibit positive LogD7.4 values that are in the range for molecules capable of crossing the cell membrane via passive difussion. Their biological activity against Toxoplasma gondi was investigated, and IC50 and lethal dose (LD50) values were determined. The ornidazole copper(II) compounds showed very good antiparasitic activity in its tachyzoite morphology. The interaction of the coordination compounds with DNA was examined by circular dichroism, fluorescence (using intercalating ethidium bromide and minor groove binding Hoechst 33258) and UV-Vis spectroscopy. The copper(II) compounds interact with the minor groove of the biomolecule, whereas weaker electrostatic interactions take place with the zinc(II) compounds. The spectroscopic data achieved for the two series of complexes (namely with copper(II) and zinc(II) as metal center) agree with the respective DNA-damage features observed by gel electrophoresis.

STUDY OF HYBRIDIZATION OF COMPLEMENTARY SINGLE-STRANDED POLYNUCLEOTIDES POLY(rA) AND POLY(rU)

The study of the interaction of intercalators ethidium bromide (EtBr), methylene blue (MB) and groove binding compound Hoechst 33258 (H33258) with single-stranded synthetic polyribonucleotides poly(rA) and poly(rU) has been carried out by the method of UV-melting, at ionic strengths of the solution 0.04 M and 0.1 M. Stirring of poly(rA) and poly(rU) by equal-molar concentrations was shown to result in hybridization with formation of double-stranded (ds-) structure poly(rA)-poly(rU). It was revealed that EtBr, with higher degree in comparison with MB, stimulates hybridization and stabilizes the formed ds-structure poly(rA)-poly(rU). It was also found out that the hybridization process and affinity of EtBr and MB depend on the ionic strength of the solution and these processes occur much more effectively at the ionic strength 0.04 M. On the other hand, it was shown that the groove binding ligand H33258 practically does not affect the stabilization of the formed ds-structure poly(rA)-poly(rU).

Fluorometric Determination of DNA Nanostructure Biostability.

The analysis and improvement of DNA nanostructure biostability is one of the keys areas of progress needed in DNA nanotechnology applications. Here, we present a plate-compatible fluorometric assay for measuring DNA nanostructure biostability using the common intercalator ethidium bromide. We demonstrate the assay by testing the biostability of duplex DNA, a double crossover DNA motif, and a DNA origami nanostructure against different nucleases and in fetal bovine serum. This method scales well to measure a large number of samples using a plate reader and can complement existing methods for assessing and developing robust DNA nanostructures.

Effects of Local Sequence, Reaction Conditions, and Various Additives on the Formation and Stability of Interstrand Cross-Links Derived from the Reaction of an Abasic Site with an Adenine Residue in Duplex DNA.

The experiments described here examined the effects of reaction conditions, various additives, and local sequence on the formation and stability interstrand cross-links (ICLs) derived from the reaction of an apurinic/apyrimidinic (AP) site with the exocyclic amino group of an adenine residue on the opposing strand in duplex DNA. Cross-link formation was observed in a range of different buffers, with faster formation rates observed at pH 5. Inclusion of the base excision repair enzyme alkyladenine DNA glycosylase (hAAG) which binds tightly to AP-containing duplexes decreased, but did not completely prevent, formation of the dA-AP ICL. Formation of the dA-AP ICL was not altered by the presence of the biological metal ion Mg2+ or the biological thiol, glutathione. Several organocatalysts of imine formation did not enhance the rate of dA-AP ICL formation. Duplex length did not have a large effect on dA-AP yield, so long as the melting temperature of the duplex was not significantly below the reaction temperature (the duplex must remain hybridized for efficient ICL formation). Formation of the dA-AP ICL was examined in over 40 different sequences that varied the neighboring and opposing bases at the cross-linking site. The results indicate that ICL formation can occur in a wide variety of sequence contexts under physiological conditions. Formation of the dA-AP ICL was strongly inhibited by the aldehyde-trapping agents methoxyamine and hydralazine, by NaBH3CN, by the intercalator ethidium bromide, and by the minor groove-binding agent netropsin. ICL formation was inhibited to some extent in bicarbonate and Tris buffers. The dA-AP ICL showed substantial inherent stability under a variety of conditions and was not a substrate for AP-processing enzymes APE1 or Endo IV. Finally, we characterized cross-link formation in a small (11 bp) stem-loop (hairpin) structure and in DNA-RNA hybrid duplexes.

Anomeric DNA Strand Displacement with α-D Oligonucleotides as Invaders and Ethidium Bromide as Fluorescence Sensor for Duplexes with α/β-, β/β- and α/α-D Configuration.

DNA strand displacement is a technique to exchange one strand of a double stranded DNA by another strand (invader). It is an isothermal, enzyme free method driven by single stranded overhangs (toeholds) and is employed in DNA amplification, mismatch detection and nanotechnology. We discovered that anomeric (α/β) DNA can be used for heterochiral strand displacement. Homochiral DNA in β‐D configuration was transformed to heterochiral DNA in α‐D/β‐D configuration and further to homochiral DNA with both strands in α‐D configuration. Single stranded α‐D DNA acts as invader. Herein, new anomeric displacement systems with and without toeholds were designed. Due to their resistance against enzymatic degradation, the systems are applicable to living cells. The light‐up intercalator ethidium bromide is used as fluorescence sensor to follow the progress of displacement. Anomeric DNA displacement shows benefits over canonical DNA in view of toehold free displacement and simple detection by ethidium bromide.

Interaction of organoruthenium(II)-polypyridyl complexes with DNA and BSA.

The interaction of four arene ruthenium complexes [(η6-p-cymene)Ru(Me2dppz)Cl]PF6 (1) with Me2dppz = 11,12-dimethyldipyrido[3,2-a:2',3'-c]phenazine, [(η6-p-cymene)Ru(aip)Cl]PF6 (2) with aip = 2-(9-anthryl)-1H-imidazo[4,5-f][1,10] phenanthroline), ([(ƞ6-toluene)Ru(ppf)Cl]PF6) (3) and ([(ƞ6-p-cymene)Ru(ppf)Cl]PF6) (4) with ppf = pyrido[2',3':5,6] pyrazino[2,3-f][1,10]phenanthroline with calf thymus DNA were investigated. All of four complexes exhibit DNA-binding activity. UV-Vis spectroscopic studies revealed the intrinsic binding constants of the order 104M-1 of magnitude, indicating non-intercalative mode. Fluorescence quenching analysis showed that all complexes interfere with intercalator ethidium bromide and minor groove binder Hoechst 33258 by a singular non-intercalative mode with extent that differs by two orders of magnitude. Gel electrophoresis results on DNA cleavage assay demonstrated that all complexes produced conformational changes of supercoiled circular plasmid pUC19 in concentration dependent way. The results of fluorescence titration bovine serum albumin by 1, 2, 3 and 4 showed that all complexes significantly quench tryptophan residues fluorescence through a static quenching mechanism. The antimicrobial activity against both Gram-positive and Gram-negative bacteria analyzed. Complex 1 was most active, even on Escherichia coli was more active than positive control compound.

Synthesis, bio-physical and anti-leishmanial studies of some novel indolo[3,2-a]phenanthridine derivatives

Eight indolo[3,2-a]phenanthridine derivatives have been synthesized in a regioselective manner involving intramolecular Heck-type arylation as a key step. The compounds display interesting photophysical proprties and hence evaluated for their ability to interact with ct-DNA. Preliminary biophysical studies via UV and Fluorescence spectrophotometric titration with ct-DNA, and dye displacement studies with well known intercalator ethidium bromide and the groove binder Hoechst 33,258 reveal that the binding mode is probably minor groove binding. The prepared indolophenanthridine derivatives have also been evaluated as anti-leishmanial agents for the first time. MTT-assays for cell cytotoxicity against Leishmania promastigotes and Leishmania amastigotes were studied with the compounds 10b-f, 12–14 for the determination of their IC50 values. Cytotoxicity was determined using a murine RAW 264.7 cell line and human embryonic kidney cell line HEK 293. In L. donovani amastigote assay, compounds 10e, 10f and 12 showed good activity with relatively low cytotoxicity against RAW 264.7, resulting in acceptable selectivity indices. Selectivity index determination indicated compounds to be potent anti-leishmanial agents while 10b, 10c and 14 showed moderate selectivity index. Moreover, cell-cycle analysis of four different compounds 10b, 12, 13 and 14, representative of each group, was performed by FACS as an attempt to understand the mechanism of actions of these different sub-classes of the compounds on Leishmania.

Meso-aryl-substituted thiacarbocyanine dyes as spectral-fluorescent probes for DNA

The noncovalent interaction of meso-aryl-substituted thiacarbocyanine dyes I and II with dsDNA and ssDNA in aqueous solutions has been studied by spectral-fluorescent methods. Complexation with DNA is accompanied by both aggregation of the dyes and the formation of monomeric strongly fluorescent complexes. Experiments on molecular docking of dyes I and II with dsDNA confirm the previous assumption about the possibility of the formation of complexes of different types: intercalation between base pairs and in the grooves of the double helix of the biopolymer. The possibility of intercalation of the dyes in the complex is confirmed by experiments on thermal dissociation of dsDNA in the presence of dyes I and II, as well as experiments on the interaction of the dyes with ssDNA. An increase in the melting temperatures Tm of dsDNA is obtained in the presence of I and II, similar to that observed for the classical intercalator ethidium bromide. The limits of detection and quantification of DNA, which are important for the use of the dyes as probes for DNA, have been determined. The primary photochemical processes of the dyes in complexes with ssDNA were studied by flash photolysis technique. Complexation with ssDNA hinders photoisomerization and creates favorable conditions for the dye triplet state formation. The decay kinetics of the triplet state of the dyes were monoexponential. The rate constant of quenching of the triplet state by air oxygen was estimated for dye I complexed with ssDNA and was found to be less than the diffusion-controlled limit. This is probably a consequence of the shielding effect of the complex on the triplet quenching process.

Stabilization and structural changes of 2D DNA origami by enzymatic ligation.

The low thermal stability of DNA nanostructures is the major drawback in their practical applications. Most of the DNA nanotubes/tiles and the DNA origami structures melt below 60°C due to the presence of discontinuities in the phosphate backbone (i.e., nicks) of the staple strands. In molecular biology, enzymatic ligation is commonly used to seal the nicks in the duplex DNA. However, in DNA nanotechnology, the ligation procedures are neither optimized for the DNA origami nor routinely applied to link the nicks in it. Here, we report a detailed analysis and optimization of the conditions for the enzymatic ligation of the staple strands in four types of 2D square lattice DNA origami. Our results indicated that the ligation takes overnight, efficient at 37°C rather than the usual 16°C or room temperature, and typically requires much higher concentration of T4 DNA ligase. Under the optimized conditions, up to 10 staples ligation with a maximum ligation efficiency of 55% was achieved. Also, the ligation is found to increase the thermal stability of the origami as low as 5°C to as high as 20°C, depending on the structure. Further, our studies indicated that the ligation of the staple strands influences the globular structure/planarity of the DNA origami, and the origami is more compact when the staples are ligated. The globular structure of the native and ligated origami was also found to be altered dynamically and progressively upon ethidium bromide intercalation in a concentration-dependent manner.

Caffeine modulates the intercalation of drugs on DNA: A study at the single molecule level

We use optical tweezers to characterize the ability of Caffeine (Caf) to modulate the intercalation of drugs into the DNA double-helix at the single molecule level. When previously bound to the double-helix, Caf hinders ethidium bromide (EtBr) intercalation, decreasing its effective equilibrium binding constant with DNA. The dominant mechanism of such singular ability is a direct binding of Caf to the intercalating drugs in solution, which decreases the effective concentration of such compounds available to interact with DNA. When EtBr intercalation into the DNA double-helix occurs firstly, on the other hand, the measured cooperativity between Caf molecules interacting with DNA can be modulated, a feature also correlated to the Caf-EtBr interaction in solution. The results achieved here unveil many peculiarities about the details of such interactions at the molecular level and provide new insights on the use of Caf in therapeutic applications.

Yeast grown in continuous culture systems can detect mutagens with improved sensitivity relative to the Ames test.

Continuous culture systems allow for the controlled growth of microorganisms over a long period of time. Here, we develop a novel test for mutagenicity that involves growing yeast in continuous culture systems exposed to low levels of mutagen for a period of approximately 20 days. In contrast, most microorganism-based tests for mutagenicity expose the potential mutagen to the biological reporter at a high concentration of mutagen for a short period of time. Our test improves upon the sensitivity of the well-established Ames test by at least 20-fold for each of two mutagens that act by different mechanisms (the intercalator ethidium bromide and alkylating agent methyl methanesulfonate). To conduct the tests, cultures were grown in small, inexpensive continuous culture systems in media containing (potential) mutagen, and the resulting mutagenicity of the added compound was assessed via two methods: a canavanine-based plate assay and whole genome sequencing. In the canavanine-based plate assay, we were able to detect a clear relationship between the amount of mutagen and the number of canavanine-resistant mutant colonies over a period of one to three weeks of exposure. Whole genome sequencing of yeast grown in continuous culture systems exposed to methyl methanesulfonate demonstrated that quantification of mutations is possible by identifying the number of unique variants across each strain. However, this method had lower sensitivity than the plate-based assay and failed to distinguish the different concentrations of mutagen. In conclusion, we propose that yeast grown in continuous culture systems can provide an improved and more sensitive test for mutagenicity.

Peculiar DNA partial threading intercalative ability of tetradentate copper complex based on ONO hydrazone backbone and an ancillary ligand

Multidentate copper metal complexes have been in the limelight in the area of DNA interaction studies exhibiting intercalation, groove binding and cross linking modes. Design of metal complex based on the versatile ligands decides their mode of DNA binding behavior. Based on this, a tetradentate Copper (II) complex, [Cu(L)(4,4’-bpy)], is synthesized using ONO hydrazone ligand and ancillary ligand, 4,4’-bipyridine. It is characterized by physico-chemical and UV-Visible, FTIR, Mass and EPR spectroscopic techniques. The binding pattern of the characterized complex with DNA has been assessed by UV absorption and fluorescence spectral titrations as well as viscosity studies and it has exhibited peculiar threading intercalation. The binding constant, Kb value of the synthesized complex was found to be (4.38 ± 0.09) × 104 M− 1, greater than that of the hydrazone ligand (Kb = 2.29 × 104 M−1) and lesser than the classical intercalator ethidium bromide - EtBr (Kb = 107). The fluorescence quenching assays in the presence of ethidium bromide and viscometric studies show threading intercalative mode of binding of the complex to the DNA base pairs. Molecular docking studies further supports such a binding pattern with the bipyridine ring of the complex intercalating with deoxycytosine nucleobase of DNA. ADME (Absorption, Distribution, Metabolism and Excretion) parameters of the complex and ligand were predicted to get an idea of drug likeliness and to correlate the structural properties with semi DNA intercalative pattern of the same.

Fluorescent reversible regulation of cysteamine-capped ZnSe quantum dots successively induced by photoinduced electron transfer of herring sperm DNA and intercalation binding of ethidium bromide

A fluorescent reversible regulation was studied by fluorescence spectra, ultraviolet–visible spectra in the combination of molecular docking, which based on the photoinduced electron transfer(PET) from hsDNA (herring sperm DNA) to CA (cysteamine)-capped ZnSe QDs (quantum dots) and intercalation of ethidium bromide (EB) into hsDNA. It was proven that the QDs bound with the adding hsDNA by electrostatic force and formed 1:1 hsDNA-QDs complexes, leading to the PET from hsDNA to QDs, and consequently the fluorescence quenching of the QDs; with EB being added in the complex solution, it bound with hsDNA by intercalation interaction and caused hsDNA releasing from hsDNA-QDs complex with forming 2.5:1 EB-hsDNA complex, leading to the recovery of fluorescence, based on the greater binding constant (1.74 × 106 L·mol−1) of hsDNA with the embedded EB comparing to that of QDs with the captured hsDNA (4.25 × 104 L·mol−1). A good linear relationship existed between the fluorescence recovery yield and the EB concentrations under the range of 1.0—12.0 × 10−6 mol·L−1 with bare interference of related substances. This work provided some useful insights into the study of binding mechanism between DNAs with their intercalators and fluorescence bi-direction regulation, and showed great potential for the determination of trace EB.

DNA binding and cleavage studies of novel Betti base substituted quaternary Cu(II) and Zn(II) phthalocyanines

Betti base substituted zinc and copper phthalocyanines (ZnBBPc and CuBBPc) and their quaternized derivatives qZnBBPc and qCuBBPc were synthesized and characterized for the first time. The photophysical quantum yield (ΦF) and singlet oxygen quantum yield (ΦΔ) of ZnBBPc and qZnBBPc were measured using comparative and 1,3-diphenylisobenzofuran (DPBF) absorption titration methods respectively. The DNA binding activities of qZnBBPc and qCuBBPc against Calf-Thymus DNA (CT-DNA) were investigated using UV–visible absorption titrations, fluorescent displacement assays with classical intercalator ethidium bromide (EB) and minor groove binder Hoechst dye (HD), as well as by carrying out viscosity measurements in Tris-NaCl buffer (pH 7.4). The results indicated that qCuBBPc and qZnBBPc phthalocyanines showed strong affinities for CT-DNA through intercalation with binding constants (Kb) of the order of 105 M−1. The cleavage activities of compounds on supercoiled pBR322 plasmid DNA were determined by agarose gel electrophoresis in the presence and absence of light and additives. The compound qCuBBPc showed hydroxyl radical mediated oxidative cleavage, while the complex qZnBBPc exhibited singlet oxygen mediated photocleavage.

STUDY OF BINDING PECULIARITIES OF ETHIDIUM BROMIDE AND METHYLENE BLUE WITH DOUBLE-STRANDED RNA

The paper presents the results of studies on the interaction of intercalators ethidium bromide (EtBr) and methylene blue (MB) with synthetic double-stranded (ds) ribonucleotide ds-poly(rA)-poly(rU) at a solution ionic strengths of 0.02, 0.04 and 0.1 M. These results revealed that poly(rA)-poly(rU) has a relatively unstable structure at the ionic strengths μ$\leq$0.02 M, which leads to the interaction of the mentioned ligands with this polynucleotide. EtBr affinity for ds-poly(rA)-poly(rU) was revealed not to depend on the ionic strength of the solution, while the affinity of MB for this polynucleotide depends on this factor, as well as on the structural state of the polynucleotide.

A Porphyrin-DNA Chiroptical Molecular Ruler With Base Pair Resolution.

DNA-based molecular rulers enable scientists to determine important parameters across biology, from the measurement of protein binding interactions, to the study of membrane dynamics in cells. However, existing rulers can suffer from poor nanometre resolution due to the flexible nature of linkers used to tether to the DNA framework. We aimed to overcome this problem using zinc and free-base porphyrin chromophores attached via short and rigid acetylene linkers. This connectivity enables the distance and angle between the porphyrins to be fine-tuned along the DNA scaffold. The porphyrins undergo favorable energy transfer and chiral exciton coupling interactions to act as highly sensitive molecular ruler probes. To validate the system, we monitored the detection of small changes in DNA structure upon intercalation of ethidium bromide. CD spectroscopy showed the porphyrins undergo highly sensitive changes in excitation coupling to facilitate base pair resolution of the novel system.