Showing papers by "Jacqueline K. Barton published in 1992"

Novel dipyridophenazine complexes of ruthenium(II): exploring luminescent reporters of DNA

Abstract: A series of ruthenium(II) complexes have been prepared which contain two phenanthroline ligands and a third bidentate ligand which is one of a set of derivatives of the parent dipyrido[3,2-a:2',3'c]phenazine (DPPZ) ligand. The spectroscopic properties of these complexes in the presence and absence of DNA have also been characterized. The derivatives have been prepared by condensation of different diaminobenzenes or diaminopyridines with the synthetic intermediate bis(1,10-phenanthroline)(1,10-phenanthroline-5,6-dione)ruthenium(II). [Ru(phen)_2DPPz](2+), like [Ru(bpy)_2DPPz]^(2+), acts as a molecular "light switch" for the presence of DNA, displaying no detectable photoluminescence in aqueous solution but luminescing brightly on binding to DNA. None of the DPPZ derivatives prepared show comparable "light switch" enhancements, since some luminescence may be detected in aqueous solution in the absence of DNA. For some complexes, however, luminescence enhancements of a factor of 20-300 are observed on binding to DNA. For these and the parent DPPZ complexes, the large enhancements observed are attributed to a sensitivity of the ruthenium-DPPZ luminescent charge-transfer excited state to quenching by water; although these complexes show little or no luminescence in water, appreciable luminescence is found in acetonitrile. Other derivatives show little solvent sensitivity in luminescence, and these, like Ru(phen)_3^(2+), display moderate enhancements (20-70%) on binding to DNA. [Ru(phen)_2DPPz]^(2+) and its derivatives all show at least biexponential decays in emission. Two binding modes have been proposed to account for these emission characteristics: a perpendicular mode where the DPPZ ligand intercalates from the major groove such that the metal-phenazine axis lies along the DNA dyad axis, and another, side-on mode where the metal-phenazine axis lies along the long axis of the base pairs.

Characterization of dipyridophenazine complexes of ruthenium(II): the light switch effect as a function of nucleic acid sequence and conformation.

Abstract: Spectroscopic parameters for two novel ruthenium complexes on binding to nucleic acids of varying sequences and conformations have been determined. These complexes, Ru(bpy)_2dppz^(2+) and Ru(phen)_2dppz^(2+) (bpy = 2,2'-bipyridine; phen = 1,10-phenanthroline; dppz = dipyrido[3,2:ɑ-2',3':c]phenazine) serve as "molecular light switches" for DNA, displaying no photoluminescence in aqueous solution but luminescing intensely in the presence of DNA. The luminescent enhancement observed upon binding is attributed to the sensitivity of the excited state to quenching by water; in DNA, the metal complex, upon intercalation into the helix, is protected from the aqueous solvent, thereby preserving the luminescence. Correlations between the extent of protection (depending upon the DNA conformation) and the luminescence parameters are observed. Indeed, the strongest luminescent enhancement is observed for intercalation into DNA conformations which afford the greatest amount of overlap with access from the major groove, such as in triple helices. Differences are observed in the luminescent parameters between the two complexes which also correlate with the level of water protection. In the presence of nucleic acids, both complexes exhibit biexponential decays in emission. Quenching studies are consistent with two intercalative binding modes for the dppz ligand from the major groove: one in which the metal-phenazine axis lies along the DNA dyad axis and another where the metal-phenazine axis lies almost perpendicular to the DNA dyad axis. Ru(bpy)_2dppz^(2+) and Ru(phen)_2dppz^(2+) are shown here to be unique reporters of nucleic acid structures and may become valuable in the design of new diagnostics for DNA.

DNA photocleavage by phenanthrenequinone diimine complexes of rhodium(III): shape-selective recognition and reaction

Abstract: Rh(phen)_2phi^(3+) and Rh(phi)_2bpy^(3+) (phi = 9,10-phenanthrenequinone diimine, phen = 1, 10-phenanthroline, bpy = 2,2’-bipyridyl) bind double helical DNA avidly (K ≥ 10^7 M^(-1)) by intercalation and with photoactivation promote strand cleavage. Rh(phen)_2phi^(3+) and Rh(phi)_2bpy^(3+) unwind double helical DNA by 21° and 18°, respectively, per bound complex. The quantum yields for nucleic acid base release at 313 nm are 0.0012 for Rh(phen)_2phi^(3+) and 0.0003 for Rh(phi)_2bpy^(3+). While both complexes have similar photochemical properties, overall binding modes and affinities, their cleavage patterns, observed on ^(32)P-end-labeled DNA restriction fragments and oligonucleotide substrates, indicate substantially different recognition characteristics. Rh(phen)_2phi^(3+) binds DNA with some sequence selectivity, preferring 5’-pyrimidine-pyrimidine-purine-3’ sites and cleaving with 5’-asymmetry, while Rh(phi)_2bpy^(3+) binds in a predominantly sequence-neutral fashion. These differences in recognition characteristics may be understood based upon the different shapes of the complexes. Owing to steric interactions of the ancillary phenanthroline ligands, Rh(phen)_2phi^(3+) appears to bind preferentially to sites which are more open in the major groove; since no similar steric constraints arise with an ancillary phi ligand, Rh(phi)_2bpy^(3+) binds all sites with similar affinities. The shapes of these complexes also govern their chemistry of strand scission. Chemical modification studies and HPLC analyses of the DNA termini and monomeric products formed in the Rh(phi)^(3+) induced DNA cleavage reactions have been conducted to characterize the products formed upon photoreaction of the rhodium complexes with 5’-CTGGCATGCCAG-3’. For Rh(phen)_2phi^(3+), the primary products are oligomers containing 3’- and 5’-phosphate termini and nucleic acid bases (in stoichiometric proportion). For Rh(phi)_2bpy^(3+), these same products account for approximately 70% of the reaction, but in addition base propenoic acids and a terminus assigned as a 3’-phosphoglycaldehyde are obtained in a correlated amount (30% of reaction). The formation of base propenoic acids and 3’-phosphoglycaldehydes are found furthermore to depend upon oxygen concentration, while other products are oxygen-independent. The products obtained are consistent with photoreaction of Rh(phi)^(3+), intercalated in the major groove of DNA, via abstraction of a C3’-H atom of the deoxyribose. Subsequent addition of dioxygen to the C3’-H radical or solvation would lead to the degradation products obtained. The partitioning between the oxygen dependent and independent pathways of DNA strand scission is found to correlate best with how the shape of the complex limits access of dioxygen to the C-3’ position. While Rh(phi)_2bpy^(3+) was found to promote the oxygen-dependent pathway to an extent of approximately 30%, Rh(phen)_2phi^(3+), with ancillary phenanthrolines that overhang and shelter the C3’-position, appears to disfavor this pathway of DNA degradation. These studies underscore the importance of shape-selection in governing not only recognition but also reaction of molecules on the helix. Such an intimate relationship between recognition and reaction of molecules bound selectively to DNA requires consideration in understanding the reactions of DNA-binding proteins and small molecules.

Transition metal complexes as probes of nucleic acids

Abstract: This chapter discusses a series of transition metal complexes that recognize the nucleic acid binding sites based on shape selection By matching the shapes and symmetries of the metal complexes to particular variations in local nucleic acid conformation, a family of molecules that target different DNA sites have been developed The recognition of a site depends on the local conformation, or shape, rather than on the sequence directly Indeed, based purely on such considerations of shape and symmetry, a high level of specificity may be achieved The molecules prepared serve as a novel series of conformation-selective probes, and these may be utilized to map the topological variations in structure along the nucleic acid polymer The chapter also discusses the sequence-neutral cleavage complex, Rh(phi)^2bpy^(3+) (where bpy is bipyridine), a useful reagent for high-resolution photofootprinting, as well as several conformation specific tools to examine nucleic acid structure These various ruthenium and rhodium complexes may be applied to detect subtle variations in B-DNA conformations or to investigate global secondary structures of a polynucleotide such as DNA cruciforms, left-handed Z-DNA, and A-form DNA The chapter describes the way the secondary and tertiary structure of RNA may be examined using this methodology The different information that may be gained from these methods is also discussed In general, studies with these complexes provide a unique and sensitive handle to probe elements of nucleic acid polymorphism in solution

Recognition of tertiary structure in tRNAs by Rh(phen)2phi3+, a new reagent for RNA structure-function mapping.

Abstract: With photoactivation Rh(phen)_2phi^(3+) promotes strand cleavage at sites of tertiary interaction in tRNA. The rhodium complex, which binds double-helical DNA by intercalation in the major groove, yields no cleavage in double-helical regions of the RNA or in unstructured single-stranded regions. Instead, Rh(phen)_2phi^(3+) appears to target regions which are structured so that the major groove is open and accessible for stacking with the complex, as occurs where bases are triply bonded. So as to examine the specificity of this novel reagent and to evaluate its use in probing structural changes in RNAs, cleavage studies have been conducted on two structurally characterized tRNAs, tRNA^(Phe) and tRNA^(Asp) from yeast, the unmodified yeast tRNA^(Phe) transcript, and a chemically modified tRNA^(phe), as well as on a series of tRNA^(Phe) mutants. On tRNA^(Phe) strong cleavage is observed at residues G22, G45, U47, ψ55, and U59; weaker cleavage is observed at A44, m^7G46, and C48. On tRNA^(AsP) cleavage is found at residues A21 through G26, ψ32, and U48, with minor cleavage apparent at A44, G45, A46, ψ55, U59, and U60. There is a striking similarity in cleavage observed on these tRNAs, and the sites of cleavage mark regions of tertiary folding. Cleavage on the unmodified tRNA^(Phe) transcript resembles closely that found on native yeast tRNA^(Phe), but additional sites, primarily in the anticodon loop and stem, are evident. The results indicate that globally the structures containing or lacking the modified bases appear to be the same; the differences in cleavage observed may reflect a loosening or alteration in the structure due to the absence of the modified bases. Cleavage results on mutants of tRNA^(Phe) illustrate Rh(phen)_2phi^(3+) as a sensitive probe in characterizing tRNA tertiary structure. Results are consistent with other assays for structural or functional changes. Uniquely, Rh- (phen)_2phi^(3+) appears to target directly sites of tertiary interaction. Cleavage results on mutants which involve base changes within the triply bonded region of the molecule indicate that it is the structure of the triply bonded array rather than the individual nucleotides which are being targeted. Chemical modification to promote selective depurination of the third base (m^7G46) involved in the triple in the folded, native tRNA leads to the reduction of cleavage by the metal complex; this result shows directly the importance of the stacked triple base structure for recognition by the metal complex. The cleavage results are consistent with the notion that Rh(phen)_2phi^(3+) preferentially targets regions of tertiary structure in the tRNA because these regions are structured so that the major grooves are open and accessible to stacking by the complex. Since sites cleaved by the rhodium complex mark a range of tertiary structures, Rh(phen)_2phi^(3+)appears to be a powerful and unique probe in characterizing the folded structures of RNAs.

Recognition of G-U mismatches by tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III)

Abstract: The coordination complex tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III) [Rh(DIP)3(3+)], which promotes RNA cleavage upon photoactivation, has been shown to target specifically guanine-uracil (G-U) mismatches in double-helical regions of folded RNAs. Photoactivated cleavage by Rh(DIP)3(3+) has been examined on a series of RNAs that contain G-U mismatches, yeast tRNA(Phe) and yeast tRNA(Asp), as well as on 5S rRNAs from Xenopus oocytes and Escherichia coli. In addition, a "microhelix" was synthesized, which consists of seven base pairs of the acceptor stem of yeast tRNA(Phe) connected by a six-nucleotide loop and contains a mismatch involving residues G4 and U69. A U4.G69 variant of this sequence was also constructed, and cleavage by Rh(DIP)3(3+) was examined. In each of these cases, specific cleavage is observed at the residue which lies to the 3'-side of the wobble-paired U; some cleavage by the rhodium complex is also evident in several structured RNA loops. The remarkable site selectivity for G-U mismatches within double-helical regions is attributed to shape-selective binding by the rhodium complex. This binding furthermore depends upon the orientation of the G-U mismatch, which produces different stacking interactions between the G-U base pair with the Watson-Crick base pair following it on the 5'-side of U compared to the Watson-Crick pair preceding it on the 3'-side of U. Rh(DIP)3(3+) therefore serves as a unique probe of G-U mismatches and may be useful both as a model and in probing RNA-protein interactions as well as in identifying G-U mismatches within double-helical regions of folded RNAs.

Nitroxide-labeled ruthenium(II)-polypyridyl complexes as EPR probes to study organized systems. 2. Combined photophysical and EPR investigations of B-DNA

Abstract: We report here the application of two polypyridyl complexes of ruthenium(II), R~(phen)~(phen-T)CI~ and Ru- (bpy),(phen-T)C12, to the examination of the interactions of the family of polypyridyl metal complexes with B-DNA. Phen-T is a modified 1,lO-phenanthroline ligand where T is a stable nitroxide (TEMPO, 2,2,6,6-tetramethylpiperidine-N-oxyl) which is covalently attached to the phenanthroline unit via a carbamate linkage. These nitroxide-substituted ruthenium complexes are unique in that the same compound is a probe which can be monitored by two completely independent spectroscopic techniques. We report here the comparison of time-resolved luminescence measurements and electron paramagnetic resonance (EPR) spectra in the presence of B-DNA and confirm that the data obtained using both methods are mutually consistent. The EPR spectra provide independent evidence for two distinct modes of binding of these complexes with DNA: one surface and the other intercalative. The EPR spectra have been evaluated to determine the rotational correlation times of motion of the bound radicals; all experimentally recorded EPR spectra have been successfully simulated.

Delineation of structural domains in eukaryotic 5S rRNA with a rhodium probe.

Abstract: The three-dimensional folding of Xenopus oocyte SS rRNA has been examined using the coordination complex Rh(phen)_2phi^(3+) (phen = phenanthroline; phi = phenanthrenequinone diimine) as a structural probe. Rh(phen)_2phi^(3+) binds neither double-helical RNA nor unstructured single-stranded regions of RNA. Instead, the complex targets through photoactivated cleavage sites of tertiary interaction which are open in the major groove and accessible to stacking. The sites targeted by the rhodium complex have been mapped on the wild-type Xenopus oocyte RNA, on a truncated RNA representing the arm of the molecule comprised of helix IV-loop E-helix V, and on several single-nucleotide mutants of the SS rRNA. On the wild-type SS rRNA, strong cleavage is found at residues U73, A74, AIOI, and U102 in the E loop and USO and G81 in helix IV; additional sites are evident at A22 and AS6 in the B loop, C29 and A32 in helix III, and C34, C39, A42, and C44 in the C loop. Given the similarity observed in cleavage between the full SS RNA and the truncated fragment as well as the absence of any long-range effects on cleavage in mutant RNAs, the results do not support models which involve long-range tertiary interactions. Cleavage results with Rh(phen)_2phi^(3+) do, however, indicate that the apposition of several noncanonical bases as well as stem-loop junctions may result in intimately stacked structures with opened major grooves. In particular, on the basis of cleavage results on mutant RNAs, both loops C and E represent structures where the strands constituting each loop are not independent of one another but are intrinsically structured. Stem-loop junctions, helix bulges containing more than one unmatched nucleotide, and a U-U mismatch also appear to provide open major grooves for targeting by Rh(phen)_2phi^(3+). These distinctive structures may also be utilized for specific recognition by proteins, such as the transcription factor TFIIIA, that bind to 5S rRNA.

Protein surface recognition and covalent binding by chromium nitrilotriacetate complexes: elucidation using NMR and CD spectroscopies

Abstract: New approaches to delineate sites of noncovalent and covalent binding by small metal complexes on a protein surface are described. The complexes employed are based on the chromium(III) nitrilotriacetate framework substituted with amino acid side chain groups. The protein examined is the well-characterized hen egg white lysozyme. Noncovalent binding location and mode have been studied by the technique of nuclear Overhauser effect quenching, along with paramagnetic relaxation experiments. These methods allow the elucidation of two major and two minor binding sites on the protein surface

Imaging of oligonucleotide-metal complexes by scanning tunneling microscopy

Abstract: Scanning tunneling microscopy (STM) has been used to image synthetic oligonucleotide duplexes alone or with an intercalatively bound metal complex with submolecular resolution. The sizes of 12 and 24 base pair (bp) oligonucleotides determined from STM images are in agreement with expected values, and images of isolated duplexes resolve the two nucleotide strands of these molecules. In addition, images of the 12-bp duplex in the presence of bis(9,lO-phenanthrenequinone diimine) (2,2’-bipyridyl)rhodium(III) exhibit a new structural feature a t 14 A from the 12-bp duplex end. This new feature corresponds well to the metal binding sites determined from DNA cleavage and molecular modeling studies. These results indicate that STM can be used to image directly transition-metal complexes bound to DNA and thus suggest that metal complexes bound specifically to biological and other macromolecules could serve as useful labels in STM structural studies.

Molecular Recognition and Chemistry in Restricted Reaction Spaces. Photophysics and Photoinduced Electron Transfer on the Surfaces of Micelles, Dendrimers, and DNA

Abstract: This Account is concerned with molecular recognition in bimolecular reactions1 that occur in restricted spaces. Bimolecular reactions of interest are photoinduced electron transfers for which the reactants are positively electronically excited metal complexes (Figure 1) and another positively charged gegenion, either a metal complex or methyl viologen (MV^(2+)) that serves as an electron acceptor. The restricted reaction spaces are the interfacial regions of anionically charged polyions such as micelles, starburst dendrimers, and DNA. Molecular recognition is concerned with how specific sites on a molecular receptor are recognized by a binding substrate. Knowledge of the underlying principles of molecular recognition is useful in diverse activities such as the design of site- and conformation-specific reagents for biomolecules, the rational design of drugs and probes of polymer structure, the design of efficient catalytic systems, the design of strategies leading to the synthesis of new materials, and the design of novel nanoscopic devices.

Chemical probes for left-handed DNA and for A-DNA; chiral metal complexes as Z-specific antitumor agents and as double strand cleavers

Abstract: This invention concerns a coordination complex and salts and optically resolved enantiomers thereof, of the formula (R) 3 --M, wherein R comprises 1,10-phenanthroline or a substituted derivative thereof, M comprises a suitable transition metal, e.g. ruthenium(II), RHODIUM(III) or cobalt(III), and R is bonded to M by a coordination bond. The complexes of this invention are useful in methods for labeling, nicking and cleaving DNA. The lambda enantiomer of complexes of this invention is useful in methods for specifically labeling, detecting, nicking and cleaving Z-DNA or A-DNA. The complexes may also be used in a method for killing tumor cells and may be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition for the treatment of tumor cells in a subject. The invention further concerns methods for treating a subject affilicted with tumor cells.

Metallointercalators: structure of rac-bis(ethylenediamine)(9,10-phenanthrenequinone diimine)rhodium(III) tribromide trihydrate

Abstract: Bis(ethylenediamine)(phenanthrenequinone diimine)rhodium(III) tribromide trihydrate has a nearly planar phenanthrenequinone diimine ligand and two ordered ethylenediamine ligands, giving a distorted octahedral coordination to the compound. The N-Rh-N angles range from 77.1 (6) to 96.5°, with the largest deviations from 90° associated with the N atoms of the phenanthrenequinone diimine ligand. The two Rh-N distances involving these N atoms are 0.05 A shorter than Rh-N distances to ethylenediamine ligands; other distances and angles are within normal ranges.