RESUMO
A series of peptides containing histidine residues were designed as potential hybridization rate enhancers within a polymeric matrix of DNA microarrays. The polymeric matrix modified with these peptides showed strong attraction to DNA molecules under conditions of induction. DNA probes on the peptide-modified sites rapidly hybridized to their complementary targets with single base pair mismatch discrimination.
Assuntos
Hibridização de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/química , DNA/genética , DNA/metabolismo , Sondas de DNA , Expressão Gênica , Histidina/química , Concentração de Íons de Hidrogênio , Especificidade por SubstratoRESUMO
Photochemical attachment of synthetic oligonucleotides on the three dimensional surface of a polyacrylamide based hydrogel was used in the specific detection of target oligonucleotides. Covalent attachment of the oligonucleotide to the hydrogel was mediated by the incorporation of a 2 + 2 photo-attachable functional group in both the hydrogel and the oligonucleotide probe. Expression and SNP assays were used to evaluate this platform.
Assuntos
Hidrogéis/química , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos/química , Resinas Acrílicas/química , Humanos , Sondas de Oligonucleotídeos/química , FotoquímicaRESUMO
The 1H NMR spectrum of the tetradeoxynucleotide d(TpCpGpA) was examined as a function of temperature, pH, and concentration. At pH 7 and above the solution conformation for this oligodeoxynucleotide appears to be a mixture of random coil and Watson-Crick duplex. At 25 degrees C, a pH titration of d(TpCpGpA) shows that distinct conformational changes occur as the pH is lowered below 7.0. These conformational changes are reversible upon readjusting the pH to neutrality, indicating the presence of a pH-dependent set of conformational equilibria. At 25 degrees C, the various conformational states in the mixture are in rapid exchange on the NMR time scale. Examination of the titration curve shows the presence of distinct conformational states at pH greater than 7, and between pH 4 and pH 5. At pH less than 4, a third conformational state is present. When the pH titration is repeated at 5 degrees C, the conformational equilibria are in slow exchange on the NMR time scale; distinct signals from each conformational state are observable. The stable conformational state present between pH 4 and pH 5 represents an ordered conformation of d(TpCpGpA) which dissociates to a less ordered structure upon raising the temperature. This ordered conformation does not result from an intramolecular rearrangement, as is shown by by spectra obtained by varying oligodeoxynucleotide concentration at constant pH. The ordered conformation differs from the Watson-Crick helix, as is shown from nuclear Overhauser enhancement experiments, as well as chemical shift data. An ordered conformation for d(TpCpGpA) was previously reported [Reid, D. G., Salisbury, S. A., Brown, T., & Williams, D. H. (1985) Biochemistry 24, 4325-4332].(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Oligodesoxirribonucleotídeos , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , Soluções , TemperaturaRESUMO
Oligodeoxynucleotides are reversibly deuteriated at the purine C8 and cytosine C5 positions with deuterioammonium bisulfite at pD 7.8. The exchange reaction is complete after 48 h at 65 degrees C. When an oligomer deuteriated under these conditions is analyzed by 1H nuclear magnetic resonance (NMR) spectroscopy, the purine H8 and cytosine H5 proton signals are selectively removed from the spectrum. A non-self-complementary oligodeoxynucleotide that has been deuteriated in this manner may be annealed with its complement and the resulting heteroduplex analyzed by two-dimensional nuclear Overhauser enhancement (NOESY) spectroscopy. NOE cross-peaks arising from pyrimidine H6-deoxyribose H1' dipolar interactions in both strands are observed, but purine H8-deoxyribose H1' and purine H8-deoxyribose H2',H2" dipolar interactions are only observed for the nondeuteriated strand. The intense cytosine H5-H6 cross-peaks are also removed from the spectrum of the deuteriated strand, which further simplifies interpretation since these strong cross-peaks often interfere with less intense NOE cross-peaks arising from dipolar coupling between purine H8 or pyrimidine H6 and deoxyribose anomeric protons. The resulting spectral simplification allows unambiguous assignments to be made on NOEs that otherwise may be difficult to distinguish. The deuteration procedure is demonstrated with the sequence d(CGTTATAATGCG).d(CGCATTATAACG), which has previously been assigned by traditional NOESY methods [Wemmer, D. E., Chou, S.-H., Hare, D. R., & Reid, B. R. (1984) Biochemistry 23, 2262-2268]. Although the assignment of this dodecadeoxynucleotide may be completed without deuteriation, several NOEs must be assigned indirectly because of degeneracies in the chemical shift of the purine H8 protons.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Oligodesoxirribonucleotídeos , Composição de Bases , Sequência de Bases , Deutério , Espectroscopia de Ressonância Magnética/métodos , Conformação de Ácido NucleicoRESUMO
Certain 6-halo-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepines were found to be potent D-1 dopamine agonists. The 1-(4-hydroxyphenyl) analogues did not have central nervous system activity because their high polarity inhibited entry into the brain. However, these compounds were potent renal vasodilators. Fenoldopam, the 6-chloro analogue, is an especially significant member of the series, and its synthesis, pharmacology, and clinical properties have been studied extensively. The 6-methyl and 6-iodo congeners were potent renal vasodilators, but nonpotent partial D-1 agonists as measured by stimulation of rat caudate adenylate cyclase. A possible rationalization suggests different receptor reserves for these activities. The 9-substituted benzazepines were either inactive or of low potency as dopamine agonists, while the N-methyl analogues had significant antagonist potency as measured by inhibition of dopamine stimulation of rat caudate adenylate cyclase.
Assuntos
Benzazepinas/farmacologia , Receptores Dopaminérgicos/efeitos dos fármacos , Circulação Renal/efeitos dos fármacos , Vasodilatadores/síntese química , Adenilil Ciclases/análise , Animais , Pressão Sanguínea/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Cães , Fenoldopam , Relação Estrutura-Atividade , Vasodilatadores/farmacologiaRESUMO
A new polymer supported synthesis of deoxyribooligonucleotides which can be adapted to automation is described. The method is based on the elongation of an oligomer chain in the 5'- to 3' -end direction using the modified phosphotriester chemistry. The approach is exemplified by the synthesis of a nonanucleotide d(TTCGTCTTG).