349 research outputs found

    Template-Directed Ligation of Tethered Mononucleotides by T4 DNA Ligase for Kinase Ribozyme Selection

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    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

    Measurement of regional cerebral blood flow using ultrafast computed tomography. Theoretical aspects.

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    Herzkrankheiten und Gestation

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    Fragen und Auskünfte

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    Measuring surface forces in aqueous electrolyte solution with the atomic force microscope

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    Surface forces determine the behaviour and properties of colloids, including biological molecules, micelles and membranes. Recently it has been realized that the atomic force microscope, which is normally used to image the topography of surfaces with high resolution, can also be used to measure surface forces. The advantages of the atomic force microscope are that virtually any surface of interest can be investigated and that measurements are relatively fast and easy to perform. Furthermore, since the interacting areas are small (typically 102–1002 nm2) samples only need to be smooth and homogeneous on a small scale. Local surface properties, like the surface charge density or micromechemical properties, can be determined
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