RESUMO
The DNA chip that immobilizes DNA oligonucleotides on a solid plate surface is used for many diagnostic applications. For maximizing the detection sensitivity and accuracy, it is important to control the DNA density on a chip surface and establish a convenient method for optimizing the density. Here, the binding of DNA mismatch-binding protein MutS to the DNA substrate on the chip was investigated, which can be applied for high-throughput single-nucleotide polymorphism analysis in a genome. We prepared the DNA chips where the DNA substrate density was changed simply by using a mixed DNA solution. The binding of MutS was significantly influenced by the amount of DNA substrate on the chip as a consequence of steric crowding, and the moderate density that gave the distance between the DNA substrates greater than the size of the protein was appropriate to obtain accurate kinetic parameters. The substrate density-controlled DNA chip prepared using the mixed DNA solution has distinctive advantages for maximizing the detection capability and kinetic analysis of the binding of MutS and probably also other large proteins.
Assuntos
DNA/metabolismo , Ensaios de Triagem em Larga Escala , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oligonucleotídeos/metabolismo , Ligação Proteica/genética , Pareamento Incorreto de Bases , DNA/química , Análise Mutacional de DNA/métodos , Cinética , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Oligonucleotídeos/química , Sensibilidade e Especificidade , Gravidade Específica , Ressonância de Plasmônio de Superfície , Thermus/químicaRESUMO
To understand the influences of the ribonucleotide on a duplex conformation and its stability, we systematically studied the CD spectra and the thermodynamics of nucleic acid duplexes formed by the chimeric RNA-DNA strand in which ribonucleotides and deoxyribonucleotides were covalently attached. It was found that the duplex stability was context-dependent and independent of the number of ribonucleotides in the chimeric strand, whereas the CD spectra showed less overall structural perturbation by the chimeric junctions. Combining the results of the CD and the thermodynamic data revealed a stability-structure relationship for the duplexes. Importantly, DeltaG(o)37 values estimated for the chimeric junction formation in the RNA-DNA/DNA and the RNA-DNA/RNA duplexes were close to those of RNA/DNA and RNA/RNA interactions, respectively. Furthermore, DeltaG(o)37s of the DNA-RNA/DNA and DNA-RNA/DNA-RNA junctions were similar to those of the DNA duplex, and the values of DNA-RNA/RNA-DNA were similar to those of the DNA/RNA. The thermodynamic analyses suggest that the 5'-nucleotide may be the crucial factor that determines the stability at the chimeric junction. Our results not only suggest influences of the ribonucleotide on a duplex conformation and its stability but also are useful for the design of RNA-DNA chimeric strands applicable to biotechnology.
Assuntos
DNA/química , Ácidos Nucleicos Heteroduplexes/química , RNA/química , Ribonucleotídeos/química , Pareamento de Bases , Quimera , Dicroísmo Circular , Temperatura Alta , Conformação de Ácido Nucleico , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , TermodinâmicaRESUMO
The relative electron density resolution was discussed by the Wiener spectrum in the heavy ion CT image. The two-dimensional (2D) Wiener spectrum in the CT image was obtained from the one-dimensional (1D) Wiener spectrum of the measured residual range distribution of the water phantom for a single projection angle, and the relative electron density resolution in the CT image was calculated from the 2D Wiener spectrum. To examine the usefulness of this method, the relative electron density resolution was also estimated by other two methods; the calculation using the Wiener spectrum of the reconstructed image of the water phantom, and the estimation by the reconstructed image of the electron density resolution phantom. The result of the first method was similar to those of the other two methods. Therefore, it is useful to estimate the relative electron density resolution by the 1D Wiener spectrum of the measured residual range distribution of the water phantom for a single projection angle.
Assuntos
Elétrons , Íons Pesados , Processamento de Imagem Assistida por Computador , Imagens de Fantasmas , Tomografia Computadorizada por Raios XRESUMO
Thermodynamic parameters of RNA/DNA chimeric duplexes were obtained systematically. Stability of the duplexes including chimeric junctions was context dependent, although CD spectra showed less structural perturbation by the junctions. Nearest-neighbor delta G degree 37 values for these chimeric RNA-DNA junctions suggested that 2'-OH on the 5'-side nucleotide at the junction may be the determinant for the nearest-neighbor stability of the chimeric junction.