The structure of drug-deoxydinucleoside phosphate complex; generalized conformational behavior of intercalation complexes with RNA and DNA fragments.

A 2:2 complex of proflavine and deoxycytidylyl-3', 5'-guanosine has been crystallized and its structure determined by x-ray crystallography. The two dinucleoside phosphate strands form self complementary duplexes with Watson Crick hydrogen bonds. One proflavin is asymmetrically intercalated between the base pairs and the other is stacked above them. The conformations of the nucleotides are unusual in that one strand has C3',C2'endomixed sugar puckering and the other has C3',C3' endo deoxyribose sugars. These results show that the conformation of the 3'sugar is of secondary importance to the intercalated geometry.     

Studies of the optical properties of the proflavine–DNA complex

We report studies of the optical properties of the proflavine–DNA complex, using absorbance and circular dichroism spectroscopy. From comparison of the absorption spectra of proflavine complexed with calf thymus and T2 DNA, we conclude that stacking of the dyes external to the double helix is comparatively much weaker with T2 DXA, probably because of its glucosylation. Several sources are found for the circular dichroism induced in proflavine when it is complexed with DNA. There is a relatively weak circular dichroism induced when the dye is infinitely dilute on the DNA lattice; this presumably arises from the environmental asymmetry of the binding site. Stronger circular dichroism effects are induced by interaction of intercalated and stacked dyes; studies with T2 DNA, for which stacking seems to be blocked, permit a tentative resolution of effects due to the two modes of binding. One recurring theme of these studies is the observation that the optical properties are quite dependent on environment. The most dramatic example is a strong variation with salt concentration of the amplitude of the circular dichroism induced in the isolated (intercalated) monomer by the surrounding DNA. This suggests that the structure of the intercalated complex is quite sensitive to external conditions.     

A temperature-jump kinetic study of the binding of proflavine to poly I-poly C

The kinetics of interaction between proflavine and poly I.poly C at 25°C, neutral pH, and moderate ionic strength have been studied by relaxation methods. The qualitative features of this system resemble those previously reported by Crothers and co-workers for proflavine–DNA and proflavine–poly A·poly U interactions–two relaxations are observed coresponding to a fast bimolecular step followed by a slower isomerization. These results can best be accommodated by a two-step mechanism leading from the free dye through an “outside-bound” complex to the intercalated complex. Quantitative comparison of the various rate constants for proflavine binding to different double-helical polynucleotides shows that the rates are slower for both ribohomopolymer pairs than for DNA. The rates for poly I·poly C are approximately three times faster than these for poly A·poly U.     

Geometry of DNA–dye intercalation complexes from the study of linear dichroic spectra of stretched films

Films of DNA–dye complexes were combined with films of pure DNA deposited on poly(vinyl alcohol) support and stretched. Reproducible dichroic spectra were obtained after equilibration of the stretched films at 93% relative humidity. Dye diffusion into the supporting poly(vinl alcohol) matrix was eliminated. The long axis of intercalated acriflavine is perpendicular to the DNA helix; proflavine deviates slightly and 9-aminoacridine significantly from such an intercalation geometry. The dichroism of two mutually perpendicularly polarized transitions of 9-aminoacridine enabled us to determine both the angles of tilt and twist of the plane of the dye relative to the DNA helix in the complex.     

Effect of DNA base composition on the intercalation of proflavine. A kinetic study.

The effect of DNA base composition on the kinetics of the association between DNA and proflavine has been investigated using the temperature jump relaxation method. It is found that, regardless of the G + C base composition the results fit a two step mechanism, the second of which exhibits characteristics of intercalation of proflavine into DNA. However, they two equilibrium constants corresponding to these steps, KI and KII, depend on the nature of the DNAs. The constant KI is found to be an order of magnitude greater for M. lysodeikticus DNA (72% G + C) than for calf thymus DNA (48% G + C). Increasing G-C content thus appears to favor the intermediate non-intercalated complex of proflavine with DNA. Methylation of M. lysodeikticus DNA with dimethyl sulfate, preferentially yielding N7 methyl guanine as the modified base, again leads to an apparent two step mechanism, with the value of KI unchanged with respect to untreated DNA, while the affinity of proflavine for the intercalated complex measured by the value of KII increases for methylated DNA.     

Dielectric behavior of DNA-proflavine complex

The dielectric relaxation of namtive DNA and DNA–proflavine complexes at different DNA phosphate (P) to dye (D) ratios, were investigated in the frequency range 100 c/sec to 100 Kc/sec. The proflavine molecules were found to have a profound effect on the static dielectric constant and the relaxation time of the polymers. The static dielectric constant was oberserved to decrese with increasing level of added proflavine. At P/D = 1, the variation of dielectric constant with frequency was small. Relaxation time (τ) was greater for the DNA–proflavine complexes compared to that for free DNA, Maximum value of the relaxation time was obtained at P/D = 10. The increase in the relaxation time and decrease in the static dielectric constant were attributed to the increase in length and meutralization of surface charges of the DNA molecules, respectively, as aresult of proflavine binding.     

The induced circular dichroism of proflavine intercalated to DNA. Dye-polymer exciton interactions

The circular dichroism induced in the visible absorption band of proflavine cation isolatedly intercalated to DNA was investigated in terms of the dye-DNA base pair exciton interaction. The remarkable ionic strength dependence of the induced CD magnitude was in good accord with the CD magnitude calculated on the basis of the dye-polymer Frenkel exciton interaction model and under the extent of helix deformation required for intercalation. In particular the application of the internal and modified intercalation models coupled with the deep trap approximation implied that the preference of the modified intercalation due to electrostatic interaction between the acridine-nitrogen atom and the DNA phosphate group is combined with relatively high ionic strength compared with the internal intercalation.     

Peculiarities of DNA-proflavine binding under different concentration ratios

Binding of proflavine to calf thymus DNA is investigated by differential scanning calorimetry and spectrophotometry. It is shown that proflavine interacts with DNA by three binding modes. At high DNA—ligand concentration ratios (P/D), proflavine prefers to intercalate into GC-sites but can also insert into other sites. At low P/D ratios, proflavine interacts with DNA by the external binding mode. The parameters of proflavine-DNA complexation have been calculated from spectrophotometric concentration dependences. Thermodynamic parameters of DNA melting have been calculated from differential scanning calorimetry data.     

Proflavine Uptake and Release in Sensitive and Resistant Escherichia coli

Both Escherichia coli B and a proflavine-resistant mutant, E. coli B/Pr, took up the same amounts of proflavine when suspended in buffer containing the dye. In growth media, however, sensitive cells took up more proflavine than did resistant cells. Adding growth media or any one of several constituents of these media, including amino acids, glycerol, pyruvic acid, and metabolizable sugars, to resistant cells that had taken up proflavine in buffer caused them to lose the dye, but had less or no effect on sensitive cells. Certian salts caused an equal release of proflavine from resistant and sensitive cells. Proflavine released from resistant cells by glucose was not changed chemically. The effects of temperature and metabolic inhibitors suggest that proflavine uptake is a passive process but that its release may be an active one, dependent on metabolism. Glucose had more effect on the proflavine binding of E. coli B grown in a minimal medium than on that of cells grown in a complex medium. E. coli B was less susceptible to proflavine when growing in a minimal medium. The effects of other synthetic media on proflavine susceptibility of E. coli B were also studied. Deoxyribonucleic acid and envelopes from sensitive and resistant cells bound the same amounts of proflavine, and no difference was seen in the site of dye binding when sensitive and resistant cells that had taken up proflavine in buffer were sonically broken and fractionated. The results suggest that sensitive and resistant cells are equally permeable to proflavine but differ in the ease with which metabolites cause them to release bound proflavine. So far, however, these differences do not account completely for the ability of resistant cells to grow in high proflavine concentrations.     

Visible-light-induced OH radicals in DNA-proflavine complexes: an e.p.r. and spin trapping study

Using the spin-trapping technique, irradiation with visible light of complexes between DNA and proflavine was shown to generate OH radicals. The characteristic spectra were not obtained when proflavine or DNA were irradiated alone, nor when oxygen was absent. Using DMPO as a spin trap we found that the intensity of the DMPO-OH e.p.r. signal was enhanced when the molar ratio between bound proflavine and the DNA phosphorus increased up to a value of 0.15 demonstrating the efficiency of the intercalated dye molecules. A strong decrease of the e.p.r. signal was observed in the presence of various OH. scavengers like t-butanol, isopropanol or sodium benzoate. The OH. production was also decreased when the irradiation was made in the presence of SOD, ceruloplasmin or catalase and after addition of Chelex 100 resin.     

Relaxation kinetics of the interaction between RNA and metal-intercalators: the Poly(A).Poly(U)/platinum-proflavine system

The interactions of Poly(A).Poly(U) with the cis-platinum derivative of proflavine [{PtCl(tmen)}(2){HNC(13)H(7)(NHCH(2)CH(2))(2)}](+) (PRPt) and proflavine (PR) are investigated by spectrophotometry, spectrofluorimetry and T-jump relaxation at I=0.2M, pH 7.0, and T=25 degrees C. Base-dye interactions prevail at high RNA/dye ratio and binding isotherms analysis reveals that both dyes bind to Poly(A).Poly(U) according to the excluded site model (n=2). Only one relaxation effect is observed for the Poly(A).Poly(U)/PRPt system, whereas two effects are observed with Poly(A).Poly(U)/PR. The results agree with the sequence D+S <==> D, S <==> DS(I) <==> DS(II), where D,S is an external complex, DS(I) is a partially intercalated species, and DS(II) is the fully intercalated complex. Formation of DS(II) could be observed in the case of proflavine only. This result is interpreted by assuming that the platinum-containing residue of PRPt hinders the full intercalation of the acridine residue.     

Calculation of atom-centered partial charges for heme

Atom-centered partial charges are calculated for the Fe-heme in cytochrome P450cam for use in molecular dynamics simulations of polar substrates bound in the active site of the enzyme. Charges are fit to the electrostatic potential produced by ab initio UHF wavefunctions for an Fe-porphine model. Basis set dependence of these charges is observed using the LANL1DZ, LANL2DZ and augmented 6–31G levels of theory. Upon geometry optimization of the enzyme, these charge sets cause varying degrees of distortion of the porphyrin from its crystallographically observed conformation. Scaling the charges calculated from the augmented 6–31G basis by 75% reduces the heme distortion while preserving reasonable interactions with a polar substrate. A comparison of the calculated charges with other published values is presented.     

Molecular and crystal structure of an intercalation complex: Proflavine–cytidylyl-(3′,5′)-guanosine

The high-resolution crystal and molecular structure of a 3:2 complex of proflavine and cytidylyl-(3′,5′)-guanosine is described. The complex exhibits more than one mode of dye binding to the dinucleoside phosphate. One proflavine cation is symmetrically intercalated between the base pairs. The other proflavine cations and ones related by symmetry stack above and below the base pairs and also hydrogen bond externally to the duplex. The conformation of the CpG is most similar to A-RNA with all C(3′)-endo sugar puckering. To allow the base pairs to stretch from the normal 3.4-Å separation to a 6.8-Å separation, the torsion angles ? and χ of the guanosine are increased by about 60° from the values found in RNA. The crystal structure itself contains disordered sulfate anions and is highly solvated, with all but one water molecule involved in a continuous water–sulfate channel.     

X-ray crystallographic analysis of a ternary intercalation complex between proflavine and the dinucleoside monophosphates CpA and UpG

We report the crystal-structure analysis of a complex involving the drug proflavine and the two dinucleoside monophosphates cytidylyl-3′,5′-adenosine (CpA) and uridylyl-3′,5′-guanosine (UpG). The planar drug molecule is intercalated between C ?G and U ?A Watson-Crick base pairs, in a double-helical fragmentlike arrangement. Sugar conformations at the 3′-ends of the two strands are dissimilar. The backbone conformations fall within the ranges of values noted previously for dinucleoside intercalation complexes, and some correlations involving these are noted. The separation of the two strands and the basic twist angle of 16°, compared to other reported complexes, are indicative of sequence-dependent effects of the drug binding.     

Proflavine acts as a Rev inhibitor by targeting the high-affinity Rev binding site of the Rev responsive element of HIV-1

Rev is an essential regulatory HIV-1 protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome, activating the switch between viral latency and active viral replication. Previously, we have shown that selective incorporation of the fluorescent probe 2-aminopurine (2-AP) into a truncated form of the RRE sequence (RRE-IIB) allowed the binding of an arginine-rich peptide derived from Rev and aminoglycosides to be characterized directly by fluorescence methods. Using these fluorescence and nuclear magnetic resonance (NMR) methods, proflavine has been identified, through a limited screen of selected small heterocyclic compounds, as a specific and high-affinity RRE-IIB binder which inhibits the interaction of the Rev peptide with RRE-IIB. Direct and competitive 2-AP fluorescence binding assays reveal that there are at least two classes of proflavine binding sites on RRE-IIB: a high-affinity site that competes with the Rev peptide for binding to RRE-IIB (K(D) approximately 0.1 +/- 0.05 microM) and a weaker binding site(s) (K(D) approximately 1.1 +/- 0.05 microM). Titrations of RRE-IIB with proflavine, monitored using (1)H NMR, demonstrate that the high-affinity proflavine binding interaction occurs with a 2:1 (proflavine:RRE-IIB) stoichiometry, and NOEs observed in the NOESY spectrum of the 2:1 proflavine.RRE-IIB complex indicate that the two proflavine molecules bind specifically and close to each other within a single binding site. NOESY data further indicate that formation of the 2:1 proflavine.RRE-IIB complex stabilizes base pairing and stacking within the internal purine-rich bulge of RRE-IIB in a manner analogous to what has been observed in the Rev peptide.RRE-IIB complex. The observation that proflavine competes with Rev for binding to RRE-IIB by binding as a dimer to a single high-affinity site opens the possibility for rational drug design based on linking and modifying it and related compounds.     

Grand canonical ensemble Monte Carlo simulation of the dCpG/proflavine crystal hydrate.

The grand canonical ensemble Monte Carlo molecular simulation method is used to investigate hydration patterns in the crystal hydrate structure of the dCpG/proflavine intercalated complex. The objective of this study is to show by example that the recently advocated grand canonical ensemble simulation is a computationally efficient method for determining the positions of the hydrating water molecules in protein and nucleic acid structures. A detailed molecular simulation convergence analysis and an analogous comparison of the theoretical results with experiments clearly show that the grand ensemble simulations can be far more advantageous than the comparable canonical ensemble simulations.     

Mutagen–nucleic acid complexes at the polynucleotide duplex level in solution: Intercalation of proflavine into poly(dA-dT) and the melting transition of the complex

The nmr chemical shifts and line widths of the nucleic acid base and sugar proton resonances and the proflavine ring protons can be monitored through the melting transition of the proflavine + poly(dA-dT) complex, phosphate/dye (P/D) ratio = 24 and 8 in 1M salt solution. The nucleic acid and mutagen protons in the complex are in fast exchange between duplex and strand states with the midpoint of the melting transition monitored at the nucleic acid resonances increasing from 72.6°C for poly(dA-dT) to 78.1°C for the P/D = 24 complex and 83.4°C for the P/D = 8 complex in 1M salt solution. The melting transition monitored by the proflavine resonances were 80.0°C for the P/D = 24 complex and 84.3°C for the P/D = 8 complex in 1M salt solution. Since the nucleic acid is in excess at high P/D ratios, the nucleic acid transitions are an average for the opening of mutagen-free and mutagen-bound base-pair regions, while the proflavine transitions monitor the melting of mutagen-bound base-pair regions. The observed 0.75 to 0.95 ppm unfield shift at all four proflavine protons on formation of the complex with poly(dA-dT) provides direct evidence for intercalation of the mutagen between base pairs of the nucleic acid duplex. We have deduced the approximate overlap geometry between the proflavine ring and nearest-neighbor base pairs at the intercalation site from a comparison between experimental proflavine complexation shifts and those calculated for various stacking orientations. The experimental chemical shift of the poly(dA-dT) adenine H-2 resonance in the duplex state in the absence and presence of proflavine suggests that intercalation occurs preferentially at dT-dA sites. The selective chemical shift changes at the sugar H-2′,2″ and H-3′ resonances of the poly(dA-dT) duplex on complex formation demonstrates changes in the sugar pucker and/or torsion angles of the sugar phosphate backbone at the intercalation site.     

Crystallographic studies of drug-nucleic acid interactions: Proflavine intercalation between the non-complementary base-pairs of cytidilyl-3′,5′-adenosine

In order to get insights into the binding of dyes and mutagens with denatured and single-stranded nucleic acids and the possible implications in frameshift mutagenesis, a 1:1 complex between the non-self-complementary dinucleoside monophosphate cytidilyl-3′,5′-adenosine (CpA) and proflavine was crystallized. The crystals belong to the tetragonal space group P4₂2₁2 with cell constants $\text{a = b = 19.38(1)}\text{A}\text{?}\text{and}\text{c = 27.10(1)}\text{A}\text{?}$. The asymmetric unit contains one CpA, one proflavine and nine water molecules by weight. The structure was determined using Patterson and direct methods and refined to an R-value of 11% using 2454 diffractometer intensities.The non-self-complementary dinucleoside monophosphate CpA forms a selfpaired parallel chain dimer with a proflavine molecule intercalated between the protonated cytosine-cytosine (C · C) pair and the neutral adenine-adenine (A · A) pair. The dimer complex exhibits a right-handed helical twist and an irregular girth. The neutral A · A pair is doubly hydrogen-bonded through the N(6) and N(7) sites (C(1′)C(1′) distance: 10.97(2) Å) and the protonated C · C pair is triply hydrogen-bonded with a proton shared between the N(3) sites (C(1′)C(1′) distance: 9.59(2) Å). To accommodate the intercalating dye, the sugars of successive nucleotide residues adopt the two fundamental conformations (5′ end: 3′-endo, 3′ end: 2′-endo), the backbone adopts torsion angle values that fluctuate within their preferred conformational domains: the PO bonds (ω, ω′) adopt the characteristic helical (gauche^?-gauche^?) conformation, the CO bonds (φ, φ′) are both in the trans domain and the C(4′)C(5′) bonds (ψ) are in the gauche⁺ region. The bases of both residues are disposed in the preferred anti domain with the glycosyl torsion angles (χ) correlated to the puckering mode of the sugar so that the cytidine residue is C(3′)-endo, low χ (12 dg), and the adenosine residue is C(2′)-endo, high χ (84 °). The intercalated proflavine stacks more extensively with the C · C pair than the A · A pair. Between 4₂-related CpA proflavine units there is a second proflavine which stacks well with both the A · A and the C · C pairs sandwiching it. Both proflavine molecules are positionally disordered. In each of its two disordered sites, the intercalated proflavine forms hydrogen-bonded interactions with only one sugar-phosphate backbone. A total of 26 water sites has been characterized of which only two are fully occupied. These hydration sites are involved in an intricate network of hydrogen bonds with both the dye and CpA and provide insights on the various modes of interactions between water molecules and between water molecules and nucleic acids.The structure of the proflavine-CpA complex shows that intercalation of planar drugs can occur between non-complementary base-pairs. This result can be relevant for understanding the strong binding of acridine dyes to denatured DNA, single-stranded RNA, and single-stranded polynucleotides. Also, the ability of proflayine to promote self-pairs of adenine and cytosine bases could provide a chemical basis for an alternative mechanism of frameshift mutagenesis.     

Quantitative analysis of fluorescence profiles of chromosomes : Influence of DNA base composition on banding

Chromosome banding has been analysed in terms of DNA content and base composition distribution along five human chromosomes. Three intercalative dyes (quinacrine, proflavine and ethidium bromide) whose fluorescence quantum yield in the presence of DNAs of different base compositions has been determined, have been used to examine the influence of base composition on the chromosome patterns. Considering that the amount of DNA as determined by the Feulgen reaction is almost constant along the chromosome arms and assuming that base composition is the only factor influencing the fluorescence of these dyes, a distribution of the A-T base pair content along the chromosomes has been calculated from the fluorescence intensity profiles. From the ratio of the intensity profiles obtained with quinacrine and proflavine, patterns showing the variation of the DNA content and of the A-T base pair content could also be obtained independently. The validity of these different approaches is discussed.     

Extrinsic Cotton effects of proflavine bound to polynucleotides

The magnitude of the Cotton effect of proflavine which is bound to RNA or to denatured DNA depends on the ratio of bound proflavine to nucleic acid base. A statistical treatment which explains this behavior has been fitted to the experimental curves and indicates that optical activity arises through interaction between two or more bound proflavine molecules. The corresponding requirement with double helical DNA is for interaction between 3–4 proflavine molecules. Although proflavine binds to denatured DNA at pH 2.8, as shown by the shift of the proflavine spectrum, the strong binding process is absent, and to this is attributed the absence of the Cotton effect at low pH. Studies on the Cotton effects of proflavine bound to poly A and poly U at neutral pH, to poly A at acid pH and to poly (A + U) allow the generalization that a relatively rigid configuration of the binding macromolecule is required for the induction of these extrinsic Cotton effects.