ABSTRACT
Oligonucleotides are widely used as effective tools to regulate gene expression and drugs for targeted gene therapy. Therefore, they are potentially useful for the treatment of viral, tumor and hereditary diseases. Therapeutic oligonucleotides include antisense oligonucleotide, small interference RNA (siRNA), Ribozyme, DNAzyme, anti-gene, CpG, decoy and aptamer. Therapeutic oligonucleotides usually carry certain modifications, such as phosphorothioates, fluoro or locked nucleic acids, to enhance the stability and specificity, and reduce the side-effects, because natural oligonucleotides have poor stability in vivo, low specificity and side effects. Now oligonucleotides are usually manufactured by chemical synthesis, with low purity and high cost. Here, we review a novel thermocyclic reaction for the amplification of oligonucleotides, referred to as Polymerase-endonuclease Amplification Reaction (PEAR) catalyzed by two thermostable enzymes. PEAR is simple, efficient, and stable. Comparing with traditional chemical synthesis, PEAR-based enzymatic production of oligonucleotides could be a robust alternative method for the large-scale production of therapeutic or non-therapeutic oligonucleotides.
ABSTRACT
Urate oxidase (Uox), an enzyme catalyzing oxidation of uric acid to allantoin, is widely used as diagnostic reagents and for treatments of uarthritis and hyperuricemia diseases. In our study, a higher Uox producer, bacterial strain OUC-1, was isolated from soil samples. The 16S rRNA gene sequence of strain OUC-1 showed 99% identity to the homologous fragments of Bacillus fastidiosus. After purification, Uox showed the optimal pH and temperature was 10.0 and 40 °C. The Km value of Uox was (0.15±0.04) mmol/L (n=5) with uric acid as the substrate. Uox activity was enhanced by Mg²⁺, and seriously inhibited by Zn²⁺ and SDS. Then the uox gene of B. fastidiosus OUC-1 was amplified and sequenced. The 3D structures of Uox, predicted with SWISS-MODEL, showed a homotetramer structure with a subunit molecular weight of 35.38 kDa. Finally, the gene coding for the B. fastidiosus Uox was successfully cloned and heterologously expressed in E. coli, which provides theoretical basis and technical support for improvement of Uox in the future.