RESUMO
The interaction of (cytosine-5)-DNA methyltransferase SsoII (M.SsoII) with double-stranded DNA was studied by means of thickness shear mode acoustic method (TSM) and gel electrophoresis. M.SsoII recognizes in double-stranded DNA the methylation site 5'-CCNGG-3' (N=A, C, G, T) and methylates the inner cytosine residue. M.SsoII also acts as a transcription factor via binding to the regulatory site 5'-AGGACAAATTGTCCT-3' in the promoter region of SsoII restriction-modification system. We designed three 60-mer biotinylated DNA duplexes: with the methylation site (60met), with the regulatory site (60reg), and without a specific binding site (60oct). A strong binding of M.SsoII with each one of the studied DNA immobilized on the TSM transducer has been shown. The equilibrium dissociation constants, K(D), of the M.SsoII-DNA complexes decreased in the order 60oct>60reg>60met, suggesting a higher stability of M.SsoII-60met complex in comparison with the others. The association rate constant, k(a), was also higher for 60met, while similar values were obtained for 60reg and 60oct. The difference in the kinetic parameters for 60met and 60reg suggested a possible way of coordination between the two M.SsoII functions in a cell.
Assuntos
Acústica/instrumentação , DNA-Citosina Metilases/metabolismo , DNA/metabolismo , Sequência de Bases , Sítios de Ligação , Biotinilação , Citosina/química , Citosina/metabolismo , DNA/química , DNA-Citosina Metilases/química , Eletroforese em Gel de Poliacrilamida/métodos , Cinética , Metilação , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Novel affinity biosensors for detecting cellular prions, PrP(C), based on DNA aptamers and antibodies immobilized onto the carbon nanotubes have been designed and compared in accordance with their binding ability and analytical performance. The biosensors allowed us to detect PrP(C) with the limits of detection of 20 to 50 pM.
Assuntos
Anticorpos/análise , Aptâmeros de Nucleotídeos , Técnicas Biossensoriais/métodos , Proteínas PrPC/análise , Príons/análise , Proteínas Imobilizadas , Proteínas PrPC/imunologia , Príons/imunologiaRESUMO
We report on a new type of artificial receptor formed by hybridization of two DNA aptamers for human thrombin (aptabody). This aptasensor based on multiwalled carbon nanotubes allowed us to detect thrombin with detection limit of 0.3 nM, which was 3 times better in comparison with conventional aptamer.