ABSTRACT
Sensitive microplate-based chemiluminescent assay coupled with isothermal circular strand-displacement polymerization reaction (ICSDPR) for DNA detection was developed. The assay sensitivity was improved using a triple amplification strategy based on employment of ICSDPR, streptavidin-polyperoxidase conjugate and an enhanced chemiluminescent reaction. To increase the accuracy all stages of the assay (one-pot format) were carried out in the same microplate well. The proposed assay detected the target DNA in fM-pM ranges and distinguish target DNA from related mismatched DNAs, demonstrating high sensitivity and high selectivity. This format is highly suitable for the development of sensitive and high-throughput kits of nucleic acids which can be easily automated.
Subject(s)
Biosensing Techniques , DNA, Viral/analysis , Luminescent Measurements , Nucleic Acid Amplification TechniquesABSTRACT
Laccases are multicopper oxidases that catalyze oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of dioxygen to water. The physicochemical and catalytic properties of two new fungal laccases from basidiomycetes Antrodiella faginea (AfL) and Steccherinum murashkinskyi (SmL) with middle redox potential of the T1 copper site were studied. The X-ray structures of AfL and SmL were solved at 1.75â¯Å and 0.95â¯Å, respectively. The oxidized state of copper ions in the active site was observed in AfL structure, while the mixture of oxidized and reduced states was observed in SmL structure. These oxidized and reduced states relate to the position of copper ions, their coordination, and nature and position of oxygen ligands. Comparative analysis of the T1 site environment of laccases with known structure allowed us to highlight the six types of the secondary coordination sphere of the T1 copper. The solvent accessible surface area of the conservative region of the secondary coordination sphere of the T1 copper correlates with its the redox potential. It was shown that the laccase classification by the structure of the T1 copper secondary coordination sphere is in agreement to ecophysiological behavior of laccase producing fungi.
Subject(s)
Basidiomycota/enzymology , Laccase/chemistry , Protein Conformation , Catalysis , Catalytic Domain , Copper/chemistry , Crystallography, X-Ray , Ligands , Models, Molecular , Oxidation-Reduction , Oxygen/chemistry , Structure-Activity Relationship , Water/chemistryABSTRACT
This paper describes the structural analysis of the native form of laccase from Trametes hirsuta at 1.8 A resolution. This structure provides a basis for the elucidation of the mechanism of catalytic action of these ubiquitous proteins. The 1.8 A resolution native structure provided a good level of structural detail compared with many previously reported laccase structures. A brief comparison with the active sites of other laccases is given.
