RESUMO
We have developed a new method for identifying specific single- or double-stranded DNA sequences called nicking endonuclease signal amplification (NESA). A probe and target DNA anneal to create a restriction site that is recognized by a strand-specific endonuclease that cleaves the probe into two pieces leaving the target DNA intact. The target DNA can then act as a template for fresh probe and the process of hybridization, cleavage and dissociation repeats. Laser-induced fluorescence coupled with capillary electrophoresis was used to measure the probe cleavage products. The reaction is rapid; full cleavage of probe occurs within one minute under ideal conditions. The reaction is specific since it requires complete complementarity between the oligonucleotide and the template at the restriction site and sufficient complementarity overall to allow hybridization. We show that both Bacillus subtilis and B. anthracis genomic DNA can be detected and specifically differentiated from DNA of other Bacillus species. When combined with multiple displacement amplification, detection of a single copy target from less than 30 cfu is possible. This method should be applicable whenever there is a requirement to detect a specific DNA sequence. Other applications include SNP analysis and genotyping. The reaction is inherently simple to multiplex and is amenable to automation.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II , Hibridização de Ácido Nucleico/métodos , Análise de Sequência de DNA/métodos , Bacillus anthracis/genética , Bacillus subtilis/genética , DNA Bacteriano/análise , Eletroforese Capilar , Cinética , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , TemperaturaRESUMO
Conversion of the non-infectious, cellular form of the prion protein (PrP(C)) to the infectious form (PrP(Sc)) is thought to be driven by an alpha-helical to beta-sheet conformational transition. To reveal the sequence determinants which encourage the transition to beta-fold, we study the synthetic peptides associated with hydrophobic conserved fragments of the N-terminal region of the prion protein. The structure of peptides in solution was probed under various thermodynamic conditions employing circular dichroism and steady state fluorescence spectroscopy as well as dye binding assays. The fluorescence methods utilized pyrene residues covalently attached to the end of the model peptides. In aqueous solutions, the structure assessments indicate the formation of metastable peptide aggregates; the molecular conformations within the peptide micelles are largely coiled. This stage in molecular assembly exists without significant beta-strand formation, i.e., before the appearance of any ordered secondary structure detectable by circular dichroism. At moderate concentrations of trifluoroethanol and/or acetonitrile, the conformational ensemble shifts towards beta-strand formation, and the population of the amorphous aggregates decreases significantly. Overall, the present data indicate that hydrophobic interactions between side chains of the peptide variants prevent, in fact, the formation of the rigid beta-sheet structures. Encouragement of beta-folds requires the destabilization of local interactions in the peptide chain, which in vivo might be possible within cell membranes as well as within partly folded molecular forms.
Assuntos
Amiloide/química , Amiloide/fisiologia , Príons/química , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Corantes/farmacologia , Cricetinae , Relação Dose-Resposta a Droga , Humanos , Microscopia de Fluorescência , Dados de Sequência Molecular , Peptídeos/química , Doenças Priônicas/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Raios UltravioletaRESUMO
A fluorescence technology to monitor the proliferation of amyloidogenic neurological disorders is proposed. A crude brain homogenate (0.01%) from animals infected with a transmissible spongiform encephalopathy is employed as a catalytic medium initiating conformational changes in 520 nM polypeptide biosensors (Tris/trifluoroethanol 50% mixture at pH 7). The fluorescence methods utilize pyrene residues covalently attached to the peptide ends. The coil-to-beta-strand transitions in biosensor molecules cause elevation of a distinct fluorescence band of the pyrene aggregates (i.e. excimers). This approach enables the detection of infectious prion proteins at fmol, does not require antibody binding or protease treatment. Technology might be adopted for diagnosing a large variety of conformational disorders as well as for generic high-throughput screening of the amyloidogenic potential in plasma.
Assuntos
Técnicas Biossensoriais , Biotecnologia/métodos , Doenças Priônicas/metabolismo , Amiloide/química , Animais , Encéfalo/metabolismo , Dicroísmo Circular , Cricetinae , Cervos , Dimetil Sulfóxido/química , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Doenças do Sistema Nervoso/metabolismo , Peptídeos/química , Doenças Priônicas/patologia , Conformação Proteica , Estrutura Secundária de Proteína , Ovinos , Espectrometria de Fluorescência , Raios UltravioletaRESUMO
Though highly desirable, neither a single experimental technique nor a computational approach can be sufficient enough to rationalize a protein structure. The incorporation of biophysical constraints, which can be rationalized based on conventional biophysical measurements, might lead to considerable improvement of the simulation procedures. In this regard, our analysis of 180 proteins in different conformational states allows prediction of the overall protein dimension based on the chain length, i.e., the protein molecular weight, with an accuracy of 10%.