Biomedical subjects

Jonathan B Chaires

Publications and source records attributed to Jonathan B Chaires.

15 recordsLinked to original sources

PubMed2005

Molecular recognition of nucleic acids: coralyne binds strongly to poly(A).

The small molecule coralyne was found to bind preferentially and strongly to single-stranded poly(A) with an apparent association constant (Ka) of (1.8+/-0.3) x 10(6)M(-1). Binding of coralyne to poly(A) is predominantly enthalpically driven with a stoichiometry of one coralyne per four adenine bases. Poly(A) forms a coralyne dependent secondary structure with a melting temperature of 60 degrees C, for the conditions of our study.

Adenosine↗

PubMed2005

Thermal difference spectra: a specific signature for nucleic acid structures.

We show that nucleic acid structures may be conveniently and inexpensively characterized by their UV thermal difference spectra. A thermal difference spectrum (TDS) is obtained for a nucleic acid by simply recording the ultraviolet absorbance spectra of the unfolded and folded states at temperatures above and below its melting temperature (T(m)). The difference between these two spectra is the TDS. The TDS has a specific shape that is unique for each type of nucleic acid structure, a conclusion that is based on a comparison of >900 spectra from 200 different sequences. The shape of the TDS reflects the subtleties of base stacking interactions that occur uniquely within each type of nucleic acid structure. TDS provides a simple, inexpensive and rapid method to obtain structural insight into nucleic acid structures, which is applicable to both DNA and RNA from short oligomers to polynucleotides. TDS complements circular dichroism as a tool for the structural characterization of nucleic acids in solution.

DNA↗

PubMed2005

Not so crystal clear: the structure of the human telomere G-quadruplex in solution differs from that present in a crystal.

The structure of human telomere DNA is of intense interest because of its role in the biology of both cancer and aging. The sequence [5'-AGGG(TTAGGG)3] has been used as a model for telomere DNA in both NMR and X-ray crystallographic studies, the results of which show dramatically different structures. In Na+ solution, NMR revealed an antiparallel G-quadruplex structure that featured both diagonal and lateral TTA loops. Crystallographic studies in the presence of K+ revealed a flattened, propeller-shaped structure featuring a parallel-stranded G-quadruplex with symmetrical external TTA loops. We report the results of biophysical experiments in solution and computational studies that are inconsistent with the reported crystal structure, indicating that a different structure exists in K+ solutions. Sedimentation coefficients were determined experimentally in both Na+ and K+ solutions and were compared with values calculated using bead models for the reported NMR and crystal structures. Although the solution NMR structure accurately predicted the observed S-value in Na+ solution, the crystal structure predicted an S-value that differed dramatically from that experimentally observed in K+ solution. The environments of loop adenines were probed by quantitative fluorescence studies using strategic and systematic single-substitutions of 2-aminopurine for adenine bases. Both fluorescence intensity and quenching experiments in K+ yielded results at odds with quantitative predictions from the reported crystal structure. Circular dichroism and fluorescence quenching studies in the presence of the crowding agent polyethylene glycol showed dramatic changes in the quadruplex structure in K+ solutions, but not in Na+ solutions, suggesting that the crystal environment may have selected for a particular conformational form. Molecular dynamics simulations were performed to yield model structures for the K+ quadruplex form that are consistent with our biophysical results and with previously reported chemical modification studies. These models suggest that the biologically relevant structure of the human telomere quadruplex in K+ solution is not the one determined in the published crystalline state.

2-Aminopurine↗

PubMed2005

Oxazine 170 induces DNA:RNA:DNA triplex formation.

The oxazine dye (Oxazine 170) was found to induce formation of a hybrid triplex structure, poly rA:(poly dT)(2), under solution conditions in which the triplex would not otherwise form. Formation of the hybrid triplex is driven by the structural selective binding of oxazine 170 to poly rA:(poly dT)(2). Oxazine 170 serves as a lead compound for the design of new compounds that can modulate triplex formation.

Dialysis↗

PubMed2005

Competition dialysis: an assay to measure the structural selectivity of drug-nucleic acid interactions.

Competition dialysis is a powerful new tool for the discovery of ligands that bind to nucleic acids with structural- or sequence-selectivity. The method is based on firm thermodynamic principles and is simple to implement. In the competition dialysis experiment, an array of nucleic acid structures and sequences is dialyzed against a common test ligand solution. After equilibration, the amount of ligand bound to each structure or sequence is determined spectrophotometrically. Since all structures and sequences are in equilibrium with the same free ligand concentration, the amount bound is directly proportional to the ligand binding affinity. Competition dialysis thus provides a direct and quantitative measure of selectivity, and unambiguously identifies which of the structures or sequences within the sample array that are preferred by a particular ligand. Following the introduction of the method, competition dialysis has been used worldwide to probe a variety of ligand-nucleic acid interactions. This contribution will focus on new analytical approaches for extracting information from the database that resulted from the first-generation competition dialysis assay, in which binding data was gathered for the interaction of 126 compounds with 13 different structures and sequences. Such global analyses allow identification of compounds with unique types of binding selectivity.

Binding, Competitive↗

PubMed2004

Structural selectivity of aromatic diamidines.

Competition dialysis was used to study the interactions of 13 substituted aromatic diamidine compounds with 13 nucleic acid structures and sequences. The results show a striking selectivity of these compounds for the triplex structure poly dA:(poly dT)2, a novel aspect of their interaction with nucleic acids not previously described. The triplex selectivity of selected compounds was confirmed by thermal denaturation studies. Triplex selectivity was found to be modulated by the location of amidine substituents on the core phenyl-furan-phenyl ring scaffold. Molecular models were constructed to rationalize the triplex selectivity of DB359, the most selective compound in the series. Its triplex selectivity was found to arise from optimal ring stacking on base triplets, along with proper positioning of its amidine substituents to occupy the minor and the major-minor grooves of the triplex. New insights into the molecular recognition of nucleic acid structures emerged from these studies, adding to the list of available design principles for selectively targeting DNA and RNA.

Benzamidines↗

PubMed2003

Energetics of echinomycin binding to DNA.

Differential scanning calorimetry and UV thermal denaturation have been used to determine a complete thermodynamic profile for the bis-intercalative interaction of the peptide antibiotic echinomycin with DNA. The new calorimetric data are consistent with all previously published binding data, and afford the most rigorous and direct determination of the binding enthalpy possible. For the association of echinomycin with DNA, we found DeltaG degrees = -7.6 kcal mol(-1), DeltaH = +3.8 kcal mol(-1) and DeltaS = +38.9 cal mol(-1) K(-1) at 20 degrees C. The binding reaction is clearly entropically driven, a hallmark of a process that is predominantly stabilized by hydrophobic interactions, though a deeper analysis of the free energy contributions suggests that direct molecular recognition between echinomycin and DNA, mediated by hydrogen bonding and van der Waals contacts, also plays an important role in stabilizing the complex.

Animals↗

PubMed2003

Enthalpy/entropy compensation: influence of DNA flanking sequence on the binding of 7-amino actinomycin D to its primary binding site in short DNA duplexes.

The effect of the context of the flanking sequence on ligand binding to DNA oligonucleotides that contain consensus binding sites was investigated for the binding of the intercalator 7-amino actinomycin D. Seven self-complementary DNA oligomers each containing a centrally located primary binding site, 5'-A-G-C-T-3', flanked on either side by the sequences (AT)(n) or (AA)(n) (with n = 2, 3, 4) and AA(AT)(2), were studied. For different flanking sequences, (AA)(n)-series or (AT)(n)-series, differential fluorescence enhancements of the ligand due to binding were observed. Thermodynamic studies indicated that the flanking sequences not only affected DNA stability and secondary structure but also modulated ligand binding to the primary binding site. The magnitude of the ligand binding affinity to the primary site was inversely related to the sequence dependent stability. The enthalpy of ligand binding was directly measured by isothermal titration calorimetry, and this made it possible to parse the binding free energy into its energetic and entropic terms. Our results reveal a pronounced enthalpy-entropy compensation for 7-amino actinomycin D binding to this family of oligonucleotides and suggest that the DNA sequences flanking the primary binding site can strongly influence ligand recognition of specific sites on target DNA molecules.

Antineoplastic Agents↗

PubMed2003

Triplex selective 2-(2-naphthyl)quinoline compounds: origins of affinity and new design principles.

A novel competition dialysis assay was used to investigate the structural selectivity of a series of substituted 2-(2-naphthyl)quinoline compounds designed to target triplex DNA. The interaction of 14 compounds with 13 different nucleic acid sequences and structures was studied. A striking selectivity for the triplex structure poly dA:[poly dT](2) was found for the majority of compounds studied. Quantitative analysis of the competition dialysis binding data using newly developed metrics revealed that these compounds are among the most selective triplex-binding agents synthesized to date. A quantitative structure-affinity relationship (QSAR) was derived using triplex binding data for all 14 compounds used in these studies. The QSAR revealed that the primary favorable determinant of triplex binding free energy is the solvent accessible surface area. Triplex binding affinity is negatively correlated with compound electron affinity and the number of hydrogen bond donors. The QSAR provides guidelines for the design of improved triplex-binding agents.

DNA↗

PubMed2003

Biarylpyrimidines: a new class of ligand for high-order DNA recognition.

Biarylpyrimidines bearing omega-aminoalkyl substituents have been designed as ligands for high-order DNA structures: spectrophotometric, thermal and competition equilibrium dialysis assays showed that changing the functional group for substituent attachment from thioether to amide switches the structural binding preference from triplex to tetraplex DNA; the novel ligands are non-toxic and moderate inhibitors of human telomerase.

DNA↗

PubMed2003

Thermodynamic characterization of the binding of nucleotides to glycyl-tRNA synthetase.

The interaction of adenine nucleotides with glycyl-tRNA synthetase was examined by several experimental approaches. ATP and nonsubstrate ATP analogues render glycyl-tRNA synthetase more resistant to digestion by a number of proteases (thrombin, Arg-C, and chymotrypsin) at concentrations that correlate with their Michaelis constants or inhibition constants, consistent with their exerting an effect by binding at the ATP site. Glycine had little effect alone but potentiated the effect of ATP in increasing the resistance to thrombin digestion, consistent with the formation of an enzyme-bound adenylate. No protection from thrombin digestion was afforded by tRNA(gly). Binding constants were determined by isothermal titration calorimetry at 25 degrees C for ATP (2.5 x 10(5) M(-1)), AMPPNP (3.7 x 10(5) M(-1)), and AMPPCP (2.2 x 10(6) M(-1)). The nucleotides had similar values for DeltaH (-71 kJ mol(-1)), with values for TDeltaS that accounted for the differences in the binding constants. Near-ultraviolet CD spectra of the enzyme-nucleotide complexes indicate that the nucleotides are bound in the anti configuration. A glycyl-adenylate analogue, glycine sulfamoyl adenosine (GSAd), bound with a large value for DeltaH (-187 kJ mol(-1)), which was balanced by a large TDeltaS term to give a binding constant (3.7 x 10(6) M(-1)) only slightly larger than that of AMPPCP. Glycine binding to the enzyme could not be detected calorimetrically, and its presence did not change the thermodynamic parameters for binding of AMPPCP. AMPPNP and AMPPCP were not substrates for glycyl-tRNA synthetase. Analysis of the temperature dependence of ATP binding indicated that the heat capacity change is small, whereas the binding of GSAd is accompanied by a large negative heat capacity change (-2.6 kJ K(-1) mol(-1)). Titrations performed in buffers with different ionization enthalpies indicate that the large value for DeltaH for the adenylate analogue does not arise from a coupled protonation event. Differential scanning calorimetry indicated that glycyl-tRNA synthetase is stabilized by nucleotides. Unfolding of the protein is irreversible, and thermodynamic parameters for unfolding could therefore not be determined. The results are consistent with a significant conformational transition in glycyl-tRNA synthetase coupled to the binding of GSAd.

Adenosine Monophosphate↗

PubMed2003

Intercalation of trioxatriangulenium ion in DNA: binding, electron transfer, x-ray crystallography, and electronic structure.

Trioxatriangulenium ion (TOTA(+)) is a flat, somewhat hydrophobic compound that has a low-energy unoccupied molecular orbital. It binds to duplex DNA by intercalation with a preference for G-C base pairs. Irradiation of intercalated TOTA(+) causes charge (radical cation) injection that results in strand cleavage (after piperidine treatment) primarily at GG steps. The X-ray crystal structure of TOTA(+) intercalated in the hexameric duplex d[CGATCG](2) described here reveals that intercalation of TOTA(+) results in an unusually large extension of the helical rise of the DNA and that the orientation of TOTA(+) is sensitive to hydrogen-bonding interactions with backbone atoms of the DNA. Electronic structure calculations reveal no meaningful charge transfer from DNA to TOTA(+) because the lowest unoccupied molecular orbital of TOTA(+), (LUMO)(T), falls in the gap between the highest occupied molecular orbital, (HOMO)(D), and the (LUMO)(D) of the DNA bases. These calculations reveal the importance of backbone, water, and counterion interactions, which shift the energy levels of the bases and the intercalated TOTA(+) orbitals significantly. The calculations also show that the inserted TOTA(+) strongly polarizes the intercalation cavity where a sheet of excess electron density surrounds the TOTA(+).

Circular Dichroism↗

PubMed2002

Tight binding of the antitumor drug ditercalinium to quadruplex DNA.

The structural selectivity of the DNA-binding antitumor drug ditercalinium was investigated by competition dialysis with a series of nineteen different DNA substrates. The 7H-pyridocarbazole dimer was found to bind to double-stranded DNA with a preference for GC-rich species but can in addition form stable complexes with triplex and quadruplex structures. The preferential interaction of the drug with four-stranded DNA structures was independently confirmed by electrospray mass spectrometry and a detailed analysis of the binding reaction was performed by surface plasmon resonance (SPR) spectroscopy. The BIAcore SPR study showed that the kinetic parameters for the interaction of ditercalinium with the human telomeric quadruplex sequence are comparable to those measured with a duplex sequence. Slow association and dissociation were observed with both the quadruplex and duplex structures. The newly discovered preferential binding of ditercalinium to the antiparallel quadruplex sequence d(AG(3)[T(2)AG(3)](3)) provides new perspectives for the design of drugs that can bind to human telomeres.

Antineoplastic Agents↗

PubMed2002

Tiny telomere DNA.

We describe the design, synthesis and biophysical characterization of a novel DNA construct in which a folded quadruplex structure is joined to a standard double helix. Circular dichroism, gel electrophoresis, three-dimensional UV melting and differential scanning calorimetry were all used to characterize the structure. Rigorous molecular dynamics simulations were used to build a plausible atomic-level structural model of the DNA construct. This novel DNA construct provides a model for the duplex-quadruplex junction region at the end of chromosomal DNA and offers a system for the study of structure-selective ligand binding.

Base Sequence↗

PubMed2002

Daunomycin binding to deoxypolynucleotides with alternating sequences: complete thermodynamic profiles of heterogeneous binding sites.

Complete thermodynamic binding profiles for the interaction of the anticancer drug, daunomycin with natural DNA and synthetic deoxypolynucleotides were described. Fluorescence titration method was used to estimate the equilibrium binding constants. Binding isotherms were found to be surprisingly complex in some cases, presumably because there were heterogeneous sites even in simple deoxypolynucleotides of repeating sequence. Some polynucleotides consisting of alternating sequence contain at least two different binding sites for daunomycin. The binding affinity of the primary binding sites of alternating and non-alternating sequences was found to differ by two orders of magnitude. An isothermal microtitration calorimeter was used to directly measure the binding enthalpy at 25 degrees C with a high sensitivity. The binding enthalpy of poly[d(A-T)] was found to be -5.5 Kcal/mol, which was much lower than any other polynucleotides, while the binding constant of the high affinity sites, was similar. In this report, the complete thermodynamic profiles of daunomycin binding to deoxypolynucleotides were reliably shown for the first time.

Antibiotics, Antineoplastic↗