template-directed ligation of tethered mononucleotides by t4 dna ligase for kinase ribozyme selectiontemplate-directed结扎t4拴在单核苷酸的dna连接酶激酶核糖酶的选择.pdf

Template-Directed Ligation of Tethered Mononucleotides by T4 DNA Ligase for Kinase Ribozyme Selection 1,2. 3.¤ 1 1,3 David G. Nickens , Nirmala Bardiya , James T. Patterson , Donald H. Burke * 1 Department of Chemistry, Indiana University, Bloomington, Indiana, United States of America, 2 Department of Biology, Indiana University, Bloomington, Indiana, United States of America, 3 Department of Molecular Microbiology and Immunology and Department of Biochemistry, University of Missouri, Columbia, Missouri, United States of America Abstract 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 5 9 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 5 9 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: A .A .A .A .A .A .A .A and T .T .T .T T .T .T .