RESUMO
Ultraviolet thermal denaturation studies substantiate our earlier hypothesis that substitution of a L-nucleotide residue for a D-nucleotide within a DNA duplex permits a stable structure in which all bases are paired through Watson-Crick hydrogen bonds (Damha, M. J., Giannaris, P. A., Marfey, P., & Reid, L. S. (1991) Tetrahedron Lett. 32, 2573-2576). This conclusion is also evident from the NMR work of Blommers et al. [Blommers, M. J. J., et al. (1994) Biochemistry (following paper in this issue)]. Our thermal denaturation studies indicate that, while weakening the interaction with target DNA and RNA, these substitutions allow for excellent cooperative binding. When the target is single-stranded DNA, the melting temperature of the complex is lowered by 4-5 degrees C per L-dU incorporation and by 0.4-2.6 degrees C when an internal D-dC is replaced by L-dC (1 M NaCl). When the target is RNA, the depression of Tm is also greater for L-dU substitutions (5-8 degrees C) than for L-dC substitutions (2-4 degrees C). The depressions of Tm caused by introducing A/C and G/T mismatches at the same positions were significantly greater. L/D-DNA chimeras were found to activate RNase H cleavage when hybridized to RNA. Furthermore, the stability of chimeric L/D-DNA against degradation by various commercial phosphodiesterases was found to be significant, as was their stability against digestion in human serum. These experiments establish that L/D-DNA chimeras serve as excellent models of antisense oligonucleotides.
Assuntos
Oligodesoxirribonucleotídeos/química , Ribonuclease H/metabolismo , Sequência de Bases , Quimera , Ativação Enzimática , Ligação de Hidrogênio , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/síntese química , Diester Fosfórico Hidrolases/metabolismo , EstereoisomerismoRESUMO
It has been noted that 9-aminoacridine reverts a his C frameshift but not one in his D in the Salmonella strains used in the Ames test, without metabolic activation. The 2 sites differ in the arrangement of G and C residues present. We show here that a series of 9-aminoacridine derivatives exhibits the same selectivity as 9-aminoacridine provided there is at least one exocyclic amino hydrogen at the central ring position in acridines, or the analogous site in aminoquinolines. The results are consistent with a model derived from NMR experiments on 9-aminoacridine binding to dinucleoside phosphates, in which the N-H group is situated in the duplex so as to participate in a hydrogen bond with one base while excluding its complementary partner, thereby provoking mismatching. We also report a strong difference in the dose-response behavior of 9-aminoacridine, quinacrine and a bifunctional derivative of quinacrine.
Assuntos
Aminoacridinas/farmacologia , Mutagênicos , Aminoacridinas/metabolismo , Relação Dose-Resposta a Droga , Testes de Mutagenicidade , Salmonella typhimurium/genética , Relação Estrutura-AtividadeAssuntos
Hidroxilaminas/farmacologia , Mutagênicos , Animais , Bactérias , Sequência de Bases , Células Cultivadas , Aberrações Cromossômicas , Cromossomos/efeitos dos fármacos , Citosina/análogos & derivados , DNA/genética , Reparo do DNA/efeitos dos fármacos , Drosophila melanogaster/genética , Fungos/genética , Hidroxilaminas/metabolismo , Camundongos , Testes de Mutagenicidade , Plantas/genética , Ratos , Relação Estrutura-Atividade , Vírus/genéticaRESUMO
The effect of sodium bisulfite (0.27 M, pH 7) on melting behavior of DNA, yeast RNA and their respective nucleosides was studied. It was found that bisulfite added not only to pyrimidine bases but also to purine bases of nucleic acids and of nucleosides. The addition products were stable at higher temperatures but reverted to parent compounds at room temperature. The only exception was the addition product of uridine which was stable at room temperature and could be isolated by paper chromatography in a 42-62% yield. Heating of DNA solutions in the presence of bisulfite to 95 degrees C caused a 90% loss of absorbance at 260 nm. On cooling, the absorbance was essentially recovered. When compared to the melting behavior of DNA in 0.27 M NaCl or 0.09 M Na2SO4 (same ionic strength), it was found that bisulfite destabilized double helical structure of DNA and that reversible addition of bisulfite did occur much below the melting temperature of DNA observed in the other two solvents.
Assuntos
DNA , RNA , Sulfitos/farmacologia , Animais , Bovinos , Desnaturação de Ácido Nucleico/efeitos dos fármacos , Renaturação de Ácido Nucleico/efeitos dos fármacos , Nucleosídeos de Purina , Nucleosídeos de Pirimidina , Saccharomyces cerevisiae , Análise Espectral , Relação Estrutura-Atividade , TemperaturaRESUMO
The effect of chlorpromazine [2-chloro-10-(3-dimethylaminopropyl)-phenothiazine] on calf thymus DNA has been investigated by spectrophotometric, equilibrium dialysis, thermal denaturation, sedimentation and viscosity methods. The absorption spectra of DNA undergo two important changes upon binding to chlorpromazine, namely, the displacement of peaks to longer wavelength (ranging from 5-8 nm) and a decrease in the optical density. The extent of binding of chlorpromazine to native calf thymus DNA, AS MEASURED BY SPECTROPHOTOMETRIC METHOD, IS DECREASED WITH INCREASING SODIUM CHLORIDE Concentration. A curvature in the Scatchard plot suggests two types of binding processes. Chlorpromazine decreases the optical density at higher temperatures without affecting the Tm of DNA. In its presence, the absorption spectra of purine deoxynucleosides (dA, dG) and of deoxynucleotides (dAMP, dGMP) are modified, i.e., the maxima are displaced to longer wavelength (ranging from 5-17 nm) and there is a general decrease in the optical density. No such effect is observed with pyrimidine deoxynucleosides (dC, dT) and deoxy-nucleotides (dCMP, dTMP). A combination of electrostatic binding of the amino group of chlorpromazine sidechain with the negative phosphate groups of the DNA and a partial insertion of either of its two phenyl rings between the nucleotide base pairs of the DNA plus the binding caused by mutual interaction between different chlorpromazine molecules at higher concentration is proposed as a probable mode of binding of chlorpromazine to DNA.
Assuntos
Clorpromazina , DNA , Animais , Sítios de Ligação , Bovinos , Desoxirribonucleosídeos/análise , Desoxirribonucleotídeos/análise , Diálise , Temperatura Alta , Concentração de Íons de Hidrogênio , Matemática , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Solubilidade , Espectrofotometria Ultravioleta , Timo , ViscosidadeAssuntos
DNA , Dinitrofenóis , Fenil-Hidrazinas , Animais , Sítios de Ligação , Bovinos , Desoxirribonucleases , Desoxirribonucleosídeos , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Nitrobenzenos , Conformação de Ácido Nucleico , Pâncreas/enzimologia , Espectrofotometria Ultravioleta , Temperatura , TimoAssuntos
Cálcio , Colesterol , Magnésio , Acetatos , Anidridos , Sítios de Ligação , Bromo , Cloretos , Cromatografia em Camada Fina , Estabilidade de Medicamentos , Cinética , Conformação Molecular , Piridinas , Solubilidade , Solventes , Espectrofotometria Atômica , Espectrofotometria Infravermelho , Fatores de Tempo , Água , Difração de Raios XAssuntos
Bromo , Cromatina , Timo , Animais , Bovinos , Fenômenos Químicos , Química , Cromatina/análise , Cromatografia em Papel , DNA/análise , Guanidinas , Histonas/análise , Histonas/isolamento & purificação , Temperatura Alta , Hidrólise , Desnaturação de Ácido Nucleico , Nucleoproteínas/isolamento & purificação , Proteínas/análise , RNA/análise , Radiação , Espectrometria de FluorescênciaRESUMO
The effect of a water-soluble carbodiimide has been used to study the nature of the presumed anionic part of the acetylcholine (ACh) receptor at the frog neuromuscular junction. The ACh sensitivity has been measured by the moving fluid electrode method and by recording end plate potentials with microelectrodes. The carbodiimide blocked ACh sensitivity without marked effect on the membrane resistance or potential difference. The conditions of reversibility of the block and the results obtained with phospholipids suggest that a carboxyl group is important in the combination of ACh with the receptor.