Spectroscopic studies of interaction of chlorobenzylidine with DNA

Electronic absorbance and fluorescence titrations are used to probe the interaction of chlorobenzylidine with DNA. The binding of chlorobenzylidine to DNA results in hypochromism, a small shift to a longer wavelength in the absorption spectra, and emission quenching in the fluorescence spectra. These spectral characteristics suggest that chlorobenzylidine binds to DNA by an intercalative mode. This conclusion is reinforced by fluorescence polarization measurements. Scatchard plots constructed from fluorescence titration data give a binding constant of 1.3 x 10(5) M(-1) and a binding site size of 10 base pairs. This indicates that chlorobenzylidine has a high affinity with DNA. The intercalative interaction is exothermic with a Van't Hoff enthalpy of -143 kJ/mol. This result is obtained from the temperature dependence of the binding constant. The interaction of chlorobenzylidine with DNA is affected by the pH value of the solution. The binding constant has its maximum at pH 3.0. Upon binding to DNA, the fluorescence from chlorobenzylidine is quenched efficiently by the DNA bases and the fluorescence intensity tends to be constant at high concentrations of DNA when the binding is saturated. The Stern-Volmer quenching constant obtained from the linear quenching plot is 1.6 x 10(4) M(-1) at 25 degrees C. The measurements of the fluorescence lifetime and the dependence of the quenching constant on the temperature indicate that the fluorescence quenching process is static. The fluorescence lifetime of chlorobenzylidine is 1.9 +/- 0.4 ns.     

Chiral recognition by the copper(II) complex of 6-deoxy-6-N-(2-methylaminopyridine)-β-cyclodextrin

A modified β-cyclodextrin bearing a 2-aminomethylpyridine binding site for copper(II) (6-deoxy-6-[N-(2-methylamino)pyridine)]-β-cyclodextrin, CDampy was synthesized by C₆-monofunctionalization. The acid-base properties of the new ligand in aqueous solution were investigated by potentiometry and calorimetry, and its conformations as a function of pH were studied by NMR and circular dichroism (c.d.). The formation of binary copper(II) complexes was studied by potentiometry, EPR, and c.d. The copper(II) complex was used as chiral selector for the HPLC enantiomeric separation of underivatized aromatic amino acids. Enantioselectivity in the overall stability constants of the ternary complexes with D- or L-Trp was detected by potentiometry, whereas the complexes of the Ala enantiomers did not show any difference in stability. These results were consistent with a preferred cis coordination of the amino group of the ligand and of the amino acid in the ternary complexes (“cis effect”), which leads to the inclusion of the aromatic side chain of D-Trp, but not of that of L-Trp. In Trp-containing ternary complexes, the two enantiomers showed differences in the fluorescence lifetime distribution, consistent with only one conformer of D-Trp and two conformers of L-Trp, and the latter were found to be more accessible to fluorescence quenching by acrylamide and KI. Chirality 9:341–349, 1997. © 1997 Wiley-Liss, Inc.     

Pituitary somatostatin receptors. Characterization by binding with a nondegradable peptide analogue

Somatostatin receptors in the rat pituitary gland were characterized by binding analysis with a radioiodinated high affinity somatostatin analogue, 125I-Tyr1[D-Trp8]somatostatin. Receptor binding of this derivative reached equilibrium at 30 min and was maintained at a plateau for at least 60 min. Two L-Trp8- labeled somatostatin analogues. 125I-Tyr1- and [125I-Tyr11]somatostatin, displayed less stable and lower specific uptake and higher nonspecific binding. In contrast to the rapid degradation of the L-Trp8 ligands during binding assay, 125I-Tyr1]D-Trp8]somatostatin retained more than 80% of its binding activity after 90 min of incubation with pituitary particles. Pituitary particles bound 125I-Tyr1]D-Tyr8]somatostatin with high affinity (Ka = 8.6 +/- 1.2 X 10(9) M-1) and capacity of 54.4 +/- 2.6 fmol/mg. These binding sites showed specificity for the native peptide and its active analogues, and other peptide hormones, including angiotensin II, thyrotropin-releasing hormone, vasopressin, oxytocin, substance P, and gonadotropin-releasing hormone, did not inhibit tracer binding. A good correlation was observed between the binding affinities of several somatostatin analogues and their potencies as inhibitors of growth hormone release in rat pituitary cells. These findings emphasize the physiological importance of the pituitary somatostatin receptor in mediating the inhibitory action of the peptide on growth hormone release. The use of Tyr1[d-Trp8]somatostatin as a labeled ligand permits accurate determinations of the binding affinity and concentration of receptors for somatostatin in the normal pituitary gland and provides a basis for further studies of somatostatin receptor regulation and receptor-mediated cellular effects of the tetradecapeptide.     

Interactions between a symmetrical minor groove binding compound and DNA oligonucleotides: 1H and 19F NMR studies

High-resolution NMR techniques (proton and 19F) have been used to study the interactions between several DNA oligonucleotides with varying length of AT base pairs and the synthetic pyrrole-containing compound (P1-F4S-P1), which has properties similar to the DNA minor groove binding drug distamycin A. When this two-fold symmetrical DNA binding molecule is added to the self-complementary DNA oligomers, the resulting complex exhibits an NMR spectrum without any doubling of individual resonances, consistent with a two-fold symmetry of the complex. This is in contrast to all other complexes studied so far. The minimum length of an AT stretch for specific ligand binding is judged to be greater than 4 base pairs. Inter-molecular proton nuclear Overhauser effects between the ligand molecule and a DNA dodecamer d(CGCAAATTTGCG) provide evidence that P1-F4S-P1 binds DNA in the minor groove and interacts with the middle AT base pairs. The presence of a specific interaction between P1-F4S-P1 and DNA is conclusively demonstrated by 19F NMR studies, in which four previously chemically equivalent fluorine nuclei in the free molecule become two non-equivalent pairs (yielding an AB quartet pattern) upon the binding of P1-F4S-P1 to DNA duplex. A sequence-dependent binding behavior of P1-F4S-P1 is evident by comparing the 19F NMR spectra of the complexes between P1-F4S-P1 and two different but related DNA dodecamers, d(CGCAAATTTGCG) and d(CGCTTTAAAGCG). P1-F4S-P1 binds more strongly to the former dodecamer with an association constant of approximately 1 X 10(3) M-1.     

19F NMR studies of vaccinia type IB topoisomerase. Conformational dynamics of the bound DNA substrate.

The site-specific DNA cleavage and religation activities of the vaccinia virus type IB topoisomerase at (C/T)CCTT(+1)X(-1) sites in duplex DNA have allowed detailed investigations of the chemical and conformational steps on the reaction pathway of this enzyme (see accompanying article (Kwon, K., and Stivers, J. T. (2002) J. Biol. Chem. 277, 345-352)). To extend these studies to the DNA substrate, we have performed 19F NMR experiments using substrates in which the +1 T has been replaced with the NMR-sensitive thymidine base analogue 5-fluoro-2'-deoxyuridine (5-F-dUrd). Substitution of 5-F-dUrd has little effect on the binding affinity of topoisomerase I for DNA, results in small changes in the cleavage and religation rate constants, and produces a net 3-fold decrease in the cleavage equilibrium constant as compared with the CCCTT consensus DNA. One-dimensional 19F NMR experiments show that the +1 5-F-dUrd is in a dynamic equilibrium between a stacked and unstacked state in both the noncovalent complex and the covalent phosphotyrosine complex. These NMR observations are supported by the selective sensitivity of the +1 T and +1 5-F-dUrd to KMnO4 oxidation. A role for localized DNA distortion in the topoisomerase I mechanism is suggested.     

19F NMR studies of solvent exposure and peptide binding to an SH3 domain

(19)F NMR was used to study topological features of the SH3 domain of Fyn tyrosine kinase for both the free protein and a complex formed with a binding peptide. Metafluorinated tyrosine was biosynthetically incorporated into each of 5 residues of the G48M mutant of the SH3 domain (i.e. residues 8, 10, 49 and 54 in addition to a single residue in the linker region to the C-terminal polyhistidine tag). Distinct (19)F NMR resonances were observed and subsequently assigned after separately introducing single phenylalanine mutations. (19)F NMR chemical shifts were dependent on protein concentration above 0.6 mM, suggestive of dimerization via the binding site in the vicinity of the tyrosine side chains. (19)F NMR spectra of Fyn SH3 were also obtained as a function of concentration of a small peptide (2-hydroxynicotinic-NH)-Arg-Ala-Leu-Pro-Pro-Leu-Pro-diaminopropionic acid -NH(2), known to interact with the canonical polyproline II (PPII) helix binding site of the SH3 domain. Based on the (19)F chemical shifts of Tyr8, Tyr49, and Tyr54, as a function of peptide concentration, an equilibrium dissociation constant of 18 +/- 4 microM was obtained. Analysis of the line widths suggested an average exchange rate, k(ex), associated with the peptide-protein two-site exchange, of 5200 +/- 600 s(-1) at a peptide concentration where 96% of the FynSH3 protein was assumed to be bound. The extent of solvent exposure of the fluorine labels was studied by a combination of solvent isotope shifts and paramagnetic effects from dissolved oxygen. Tyr54, Tyr49, Tyr10, and Tyr8, in addition to the Tyr on the C-terminal tag, appear to be fully exposed to the solvent at the metafluoro position in the absence of binding peptide. Tyr54 and, to some extent, Tyr10 become protected from the solvent in the peptide bound state, consistent with known structural data on SH3-domain peptide complexes. These results show the potential utility of (19)F-metafluorotyrosine to probe protein-protein interactions in conjunction with paramagnetic contrast agents.     

Enzyme kinetic and spectroscopic studies of inhibitor and effector interactions with indoleamine 2,3-dioxygenase. 2. Evidence for the existence of another binding site in the enzyme for indole derivative effectors

To probe the active site of the heme protein indoleamine 2,3-dioxygenase, the effects of 3-indoleethanol (IET) (or tryptophol), one of the known indole derivative effectors, and indole (IND) on the catalytic (Vmax, Km) and spectroscopic properties (optical absorption and CD) of the enzyme were investigated. Assays were performed with the substrate L- or D-tryptophan (Trp) and an ascorbic acid-methylene blue cofactor system at 25 degrees C. This study has shown that, at millimolar concentrations, both IET and IND lower considerably the Km value for D-Trp by approximately 25% and approximately 60%, respectively, at pH 7.0, while neither affects the Km value for L-Trp. Interestingly, however, these effectors exert opposite effects with respect to each other on the Vmax values for both D-Trp and L-Trp: IET enhances the Vmax values by 40-60% while IND lowers them by 12-24%. These effects of IET and IND on the Vmax values may be attributed to the shift in the ferric (inactive) enzyme----ferrous (active) enzyme equilibrium either to the right (IET) or to the left (IND) caused by the binding of these effectors to the enzyme in the steady state of the catalytic reaction. Both effectors induce clearly detectable spectral changes, especially notable in CD spectra, upon binding (in a 1:1 molar ratio, Kd = 10(-4) to 2.5 X 10(-3) M) to the ferrous enzyme and its complexes with O2, CO, and NO, both in the presence and in the absence of L-Trp.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific recognition by the tripeptide lysyl-tryptophyl-alpha-lysine of structural damage induced in DNA by platinum derivatives

The antitumoral derivative cisPt binds to DNA, as do its inactive analogs, trans- and dienPt. Structural damage introduced into DNA after reaction with the Pt derivatives were probed by using the peptide LysTrpLys. This peptide was used for its preferential binding to single-stranded structures (Brun, F., Toulmé, J.J. and Hélène, C. (1975) Biochemistry 14, 558-563). Phosphorescence lifetime measurements show that the Pt-induced heavy atom effects are quite similar in the three peptide-DNA-Pt complexes whatever the nature of the Pt derivative used. In contrast, fluorescence quenching strongly depends on the nature of the Pt derivatives. This quenching was therefore attributed to the stacking interactions engaged by the tryptophan residue with nucleic acid bases. A comparison of fluorescence quenching data for native and modified DNAs demonstrates that modification by dienPt has no effect on stacking interactions and that high levels of modifications by trans Pt are required to observe a change in stacking efficiency. In contrast modification by cis Pt induces the formation of strong stacking sites. The results strongly suggest the existence of locally opened regions in DNA modified by cis Pt.     

Interaction of rat testis protein, TP, with nucleic acids in vitro. Fluorescence quenching, UV absorption, and thermal denaturation studies

The nucleic acid binding properties of the testis protein, TP, were studied with the help of physical techniques, namely, fluorescence quenching, UV difference absorption spectroscopy, and thermal melting. Results of quenching of tyrosine fluorescence of TP upon its binding to double-stranded and denatured rat liver nucleosome core DNA and poly(rA) suggest that the tyrosine residues of TP interact/intercalate with the bases of these nucleic acids. From the fluorescence quenching data, obtained at 50 mM NaCl concentration, the apparent association constants for binding of TP to native and denatured DNA and poly(rA) were calculated to be 4.4 X 10(3) M-1, 2.86 X 10(4) M-1, and 8.5 X 10(4) M-1, respectively. UV difference absorption spectra upon TP binding to poly(rA) and rat liver core DNA showed a TP-induced hyperchromicity at 260 nm which is suggestive of local melting of poly(rA) and DNA. The results from thermal melting studies of binding of TP to calf thymus DNA at 1 mM NaCl as well as 50 mM NaCl showed that although at 1 mM NaCl TP brings about a slight stabilization of the DNA against thermal melting, a destabilization of the DNA was observed at 50 mM NaCl. From these results it is concluded that TP, having a higher affinity for single-stranded nucleic acids, destabilizes double-stranded DNA, thus behaving like a DNA-melting protein.     

Tyrosyl-base-phenylalanyl intercalation in gene 5 protein-DNA complexes: proton nuclear magnetic resonance of selectively deuterated gene 5 protein.

The interactions of oligodeoxynucleotides with the aromatic residues of gene 5 protein in complexes with d(pA)8 and d(pT)8 have been determined by 1H NMR of the protein in which the five tyrosyl residues have been selectively deuterated either in the 2,6 or the 3,5 positions. Only the 3,5 protons of the three surface tyrosyls (26, 41, and 56) interact with the bases. The remainder of the aromatic protons undergoing base-dependent upfield ring-current shifts on complex formation are phenylalanyl protons, assigned to Phe(13) on the basis of model building. 19F NMR of the complexes of the m-fluorotyrosyl-labeled protein with d(pT)4 and d(pA)8 confirms the presence of ring-current perturbations of nuclei at the 3,5-tyrosyl positions of the three surface tyrosyls. Differential expression of the 19F(1H) nuclear Overhauser effect confirms the presence of two buried and three surface tyrosyl residues. A new model of the DNA binding groove is presented involving Tyr(26)-base-Phe(13) intercalation.     

Fluorescence determination of D- and L-tryptophan concentrations in rat plasma following administration of tryptophan enantiomers using HPLC with pre-column derivatization

Similar to L-tryptophan (L-Trp), D-Trp can be converted to unique metabolites in the mammalian body. In the present study, the difference in the plasma half-life (t(1/2)) between Trp enantiomers was investigated by following the alterations in the plasma concentration of D- or L-Trp after intraperitoneal (i.p.) administration of each enantiomer to male Sprague-Dawley rats (100 mg/kg). The investigation was performed using reversed-phase high-performance liquid chromatography (HPLC) and pre-column fluorescence derivatization with a chiral fluorescent labeling reagent, R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (R(-)-DBD-PyNCS). The t(1/2) value of D-Trp was significantly smaller than that of L-Trp, suggesting that D-Trp was eliminated from the plasma more rapidly than L-Trp. In addition, a significant increase in the plasma concentration of L-Trp was observed following administration of D-Trp, whereas no D-Trp was detected after L-Trp administration. Furthermore, the increase in the plasma concentration of L-Trp was significantly suppressed by pretreatment with an inhibitor of D-amino acid oxidase (DAAO), 3-methylpyrazole-5-carboxylic acid, which suggests that DAAO was involved in the conversion of D-Trp to L-Trp in vivo.     

Nucleic acid interactive properties of a peptide corresponding to the N-terminal zinc finger domain of HIV-1 nucleocapsid protein.

An 18-residue peptide (NC-F1) with an amino acid sequence corresponding to the N-terminal zinc finger of human immunodeficiency virus-1 nucleocapsid protein has been shown to bind to nucleic acids by fluorescence and NMR methods. Previously, this peptide has been shown to fold into a defined structure when bound to zinc (Summers et al., 1990). We have used a fluorescent polynucleotide, poly(ethenoadenylic acid), to monitor binding of this peptide to nucleic acids. In the presence of zinc, the peptide had a smaller site size (1.75 nucleotide residues/peptide) than in the absence of the metal ion (2.75). The salt sensitivity of the interaction indicated that two ion pairs are involved in the association of Zn2+ (NC-F1) with polynucleotide, whereas one ion pair is found in the metal-free peptide-nucleic acid complex. Competition experiments with single-stranded DNA (ss DNA) in either the presence or absence of Zn2+ showed that the peptide bound to ss DNA. Using NMR methods, we monitored the binding of a synthetic oligonucleotide, d(TTTGGTTT), to Zn(NC-F1). The hydrophobic residues F2 and I10, which are on the surface of the peptide and have been implicated in viral RNA recognition, were shown to interact with the oligomer. In accord with this observation, analysis of the salt dependence of the polynucleotide-peptide interaction indicates a nonelectrostatic component of about -6 kcal/mol, a value consistent with theoretical estimates of stacking energies of phenylalanine with nucleic acid bases.     

Interaction of colchicine with DNA molecules.

Colchicine (7.5 X 10-6M or 3.3 X 10-5M) was incubated at pH 7.0, 10.0, or 12.0 with high-molecular DNA from salmon sperm (7 X 10-5M or 3.5 X 10-5M DNA-P) at 40 degrees C. Interaction was monitored by UV spectrophotometry; Amax/Amin ratios, difference and additive spectra, and the quantity of colchicine bound to DNA were presented as functions of time, ionic strength and DNA concentration. Using NMR techniques, delta deltav 1/2/deltac values, chemical shifts and half-line widths of colchicine protons were analyzed as a function of the DNA/colchicine ratio, thus proving the interaction between alkaloid and DNA. An intercalation of the tropolone moiety of colchicine between the nitrogenous bases of DNA is suggested.     

Conformation and dynamics of abasic sites in DNA investigated by time-resolved fluorescence of 2-aminopurine

Abasic sites are highly mutagenic lesions in DNA that arise as intermediates in the excision repair of modified bases. These sites are generated by the action of damage-specific DNA glycosylases and are converted into downstream intermediates by the specific activity of apurinic/apyrimidinic endonucleases. Enzymes in both families have been observed in crystal structures to impose deformations on the abasic-site DNA, including DNA kinking and base flipping. On the basis of these apparent protein-induced deformations, we propose that altered conformation and dynamics of abasic sites may contribute to the specificity of these repair enzymes. Previously, measurements of the steady-state fluorescence of the adenine analogue 2-aminopurine (2AP) opposite an abasic site demonstrated that binding of divalent cations could induce a conformational change that increased the accessibility of 2AP to solute quenching [Stivers, J. T. (1998) Nucleic Acids Res. 26, 3837-44]. We have performed time-resolved fluorescence experiments to characterize the states involved in this conformational change. Interpretation of these studies is based on a recently developed model attributing the static and dynamic fluorescence quenching of 2AP in DNA to aromatic stacking and collisional interactions with neighboring bases, respectively (see the preceding paper in this issue). The time-resolved fluorescence results indicate that divalent cation binding shifts the equilibrium of the abasic site between two conformations: a "closed" state, characterized by short average fluorescence lifetime and complex decay kinetics, and an "open" state, characterized by monoexponential decay with lifetime approximately that of the free nucleoside. Because the lifetime and intensity decay kinetics of 2AP incorporated into DNA are sensitive primarily to collisional interactions with the neighboring bases, the absence of dynamic quenching in the open state strongly suggests that the fluorescent base is extrahelical in this conformation. Consistent with this interpretation, time-resolved quenching studies reveal that the open state is accessible to solute quenching by potassium iodide, but the closed state is not. Greater static quenching is observed in the abasic site when the fluorescent base is flanked by 5'- and 3'-thymines than in the context of 5'- and 3'-adenines, indicating that 2AP is more stacked with the neighboring bases in the former sequence. These results imply that the conformation of the abasic site varies in a sequence-dependent manner. Undamaged sequences in which the abasic site is replaced by thymine do not exhibit an open state and have different levels of both static and dynamic quenching than their damaged homologues. These differences in structure and dynamics may be significant determinants of the high specific affinity of repair enzymes for the abasic site.     

Fluorine-19 nuclear magnetic resonance study of codon-anticodon interaction in 5-fluorouracil-substituted E. coli transfer RNAs.

Codon-anticodon interaction was investigated in fully active 5-fluorouracil-substituted E. coli tRNAVal1 (anticodon FAC) by 19F NMR spectroscopy. Binding of the codon GpUpA results in the upfield shift of a 19F resonance at 3.9 ppm in the central region of the 19F NMR spectrum, whereas trinucleotides not complementary to the anticodon have no effect. The same 19F resonance shifts upfield upon formation of an anticodon-anticodon dimer between the 19F-labeled tRNA and E. coli tRNATyr2 (anticodon QUA). These results permit assignment of the peak at 3.9 ppm to the 5-fluorouracil at position 34 in the anticodon of fluorouracil-substituted tRNAVal1. The methionine codon ApUpG also causes a sequence-specific upfield shift of a peak in the central part of the 19F NMR spectrum of fluorinated E. coli tRNAMetm. However, ApUpG has no effect on the 19F spectrum of 19F-labeled E. coli tRNAMetf, indicating possible conformational differences between the anticodon loop of initiator and chain-elongating methionine tRNAs. 19F NMR experiments detect no binding of CpGpApA to the complementary FpFpCpG (replaces Tp psi pCpG) in the T-loop of 5-fluorouracil-substituted tRNAVal1, in the presence or absence of codon, suggesting that the tertiary interactions between the T- and D-loops are not disrupted by codon-anticodon interactions.     

Nucleotide and AP5A complexes of porcine adenylate kinase: A 1H and 19F NMR study

Proton and fluorine nuclear magnetic resonance spectroscopies (NMR) were used as methods to investigate binary complexes between porcine adenylate kinase (AK1) and its substrates. We also studied the interaction of fluorinated substrate analogues and the supposed bisubstrate analogue P1,P5-bis(5'-adenosyl) pentaphosphate (AP5A) with AK1 in the presence of Mg2+. The chemical shifts of the C8-H, C2-H, and ribose C1'-H resonances of both adenosine units in stoichiometric complexes of AK1 with AP5A in the presence of Mg2+ could be determined. The C2-H resonance of one of the adenine bases experiences a downfield shift of about 0.8 ppm on binding to the enzyme. The chemical shift of the His36 imidazole C2-H was changed in the downfield direction on ATP-Mg2+ and, to a lesser extent, AMP binding. 19F NMR chemical shifts of 9-(3-fluoro-3-deoxy-beta-D-xylofuranosyl)adenine triphosphate (3'-F-X-ATP)-Mg2+ and 9-(3-fluoro-3-deoxy-beta-D-xylofuranosyl)adenine monophosphate (3'-F-X-AMP) bound to porcine adenylate kinase could be determined. The different chemical shifts of the bound nucleotides suggest that their mode of binding is different. Free and bound 3'-F-X-AMP are in fast exchange with respect to their 19F chemical shifts, whereas free and bound 3'-F-X-ATP are in slow exchange on the NMR time scale in the absence as well as in the presence of Mg2+. This information could be used to determine the apparent dissociation constants of the nucleotides and the 3'-F-X analogues in the binary complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific recognition by the tripeptide lysyl-tryptophyl-α-lysine of structural damage induced in DNA by platinum derivatives

The antitumoral derivative cisPt binds to DNA, as do its inactive analogs, trans- and dienPt. Structural damage introduced into DNA after reaction with the Pt derivatives were probed by using the peptide LysTrpLys. This peptide was used for its preferential binding to single-stranded structures (Brun, F., Toulmé, J.J. and Hélène, C. (1975) Biochemistry 14, 558–563). Phosphorescence lifetime measurements show that the Pt-induced heavy atom effects are quite similar in the three peptide-DNA-Pt complexes whatever the nature of the Pt derivative used. In contrast, fluorescence quenching strongly depends on the nature of the Pt derivatives. This quenching was therefore attributed to the stacking interactions engaged by the tryptophan residue with nucleic acid bases. A comparison of fluorescence quenching data for native and modified DNAs demonstrates that modification by dienPt has no effect on stacking interactions and that high levels of modifications by transPt are required to observe a change in stacking efficiency. In contrast modification by cisPt induces the formation of strong stacking sites. The results strongly suggest the existence of locally opened regions in DNA modified by cisPt.     

Spectral analysis of the DNA targeting bisalkylaminoanthraquinone DRAQ5 in intact living cells.

BACKGROUND: We report on the potential DNA binding modes and spectral characteristics of the cell-permeant far red fluorescent DNA dye, DRAQ5, in solution and bound within intact cells. Our aim was to determine the constraints for its use in flow cytometry and bioimaging. METHODS: Solution characteristics and quantum yields were determined by spectroscopy. DRAQ5 binding to nuclear DNA was analyzed using fluorescence quenching of Hoechst 33342 dye, emission profiling by flow cytometry, and spectral confocal laser scanning microscopy of the complex DRAQ5 emission spectrum. Cell cycle profiling utilized an EGFP-cyclin B1 reporter as an independent marker of cell age. Molecular modeling was used to explore the modes of DNA binding. RESULTS: DRAQ5 showed a low quantum yield in solution and a spectral shift upon DNA binding, but no significant fluorescence enhancement. DRAQ5 caused a reduction in the fluorescence intensity of Hoechst 33342 in live cells prelabeled with the UV excitable dye, consistent with molecular modeling that suggests AT preference and an engagement of the minor groove. In vivo spectral analysis of DRAQ5 demonstrated shifts to longer wavelengths upon binding with DNA. Analysis of spectral windows of the dual emission peaks at 681 and 707 nm in cells showed that cell cycle compartment recognition was independent of the far red-near IR emission wavelengths monitored. CONCLUSIONS: The study provides new clues to modes of DNA binding of the modified anthraquinone molecule in vivo, and its AT base-pair selectivity. The combination of low quantum yield but high DNA affinity explains the favorable signal-to-noise profile of DRAQ5-nuclear fluorescence. The robust nature of cell cycle reporting using DRAQ5, even when restricted spectral windows are selected, facilitates the analysis of encroaching spectral emissions from other fluorescent reporters, including GFP-tagged proteins.