54 research outputs found
Template-Directed Ligation of Tethered Mononucleotides by T4 DNA Ligase for Kinase Ribozyme Selection
Background: In vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive RNA species. While nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 59 or 29 OH of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated. Methodology/Principal Findings: This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 59 phosphate. The ligation reaction therefore mimics the partition step of a selection for nucleoside kinase (deoxy)ribozymes. Ligation with tethered substrates was considerably slower than with nicked, fully duplex DNA, even though the deoxynucleotides at the ligation junction were Watson-Crick base paired in the tethered substrate. Ligation increased markedly when the bridging template strand contained unpaired spacer nucleotides across from the flexible tether, according to the trends: A2.A1.A3.A4.A0.A6.A8.A10 and T2.T3.T4.T6<T1.T8.T10. Bridging T’s generally gave higher yield of ligated product than bridging A’s. ATP concentrations above 33 mM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme. Under optimized conditions, T4 DNA ligase efficiently (.90%) joined a correctly paired, or T:G wobble-paired, substrate on the 39 side of the ligation junction while discriminating approximately 100-fold against most mispaire
Rosiglitazone pretreatment influences thrombin-induced anti-oxidative action via activating NQO1and γ-GCS in rat microglial cells
Potentiating effect of acetaminophen and carbon tetrachloride-induced hepatotoxicity is mediated by activation of receptor interaction protein in mice
Identification of functionally distinct domains of human granulocyte- macrophage colony-stimulating factor using monoclonal antibodies
Identification of functionally distinct domains of human granulocyte- macrophage colony-stimulating factor using monoclonal antibodies
Analysis of Recombinant Human IFN-Gamma Produced in CHO Cells:Effects of Glycosylation and Proteolytic Processing on Product Heterogeneity
Effects of a dietary magnesium deficiency and excess vitamin D3 on swine coronary arteries.
Short-term cigarette smoke exposure induces reversible changes in energy metabolism and cellular redox status independent of inflammatory responses in mouse lungs
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