tailieunhanh - Báo cáo khoa học: Mapping of the functional phosphate groups in the catalytic core of deoxyribozyme 10–23

The RNA phosphodiester bond cleavage activity of a series of 16 thio-de-oxyribozymes 10–23, containing a P-stereorandom single phosphorothioate linkage in predetermined positions of the catalytic core from P1 to P16, was evaluated under single-turnover conditions in the presence of either 3mmMg2+ or 3 mmMn2+ . | ễFEBS Journal Mapping of the functional phosphate groups in the catalytic core of deoxyribozyme 10-23 Barbara Nawrot Kinga Widera Marzena Wojcik Beata Rebowska Genowefa Nowak and Wojciech J. Stec Department of Bioorganic Chemistry Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences Lodz Poland Keywords catalysis deoxyribozyme phosphorothioate rescue effect thio effect Correspondence B. Nawrot Department of Bioorganic Chemistry Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences Sienkiewicza 112 90-363 Lodz Poland Fax 48 42 6815483 Tel 48 42 6816970 E-mail bnawrot@ Present address MedicalUniversity of Lodz Department of StructuralBiology Zeligowskiego Poland Received 4 October 2006 revised 29 November 2006 accepted 18 December 2006 The RNA phosphodiester bond cleavage activity of a series of 16 thio-deoxyribozymes 10-23 containing a P-stereorandom single phosphorothioate linkage in predetermined positions of the catalytic core from P1 to P16 was evaluated under single-turnover conditions in the presence of either 3 mM Mg2 or 3 mM Mn2 . A metal-specificity switch approach permitted the identification of nonbridging phosphate oxygens proRP or proSP located at seven positions of the core P2 P4 and P9-13 involved in direct coordination with a divalent metal ion s . By contrast phosphorothioates at positions P3 P6 P7 and P14-16 displayed no functional relevance in the deoxyribozyme-mediated catalysis. Interestingly phosphorothioate modifications at positions P1 or P8 enhanced the catalytic efficiency of the enzyme. Among the tested deoxyribozymes thio-substitution at position P5 had the largest deleterious effect on the catalytic rate in the presence of Mg2 and this was reversed in the presence of Mn2 . Further experiments with thio-deoxyribozymes of stereodefined P-chirality suggested direct involvement of both oxygens of the P5 phosphate and the proRP oxygen at P9 in the metal ion coordination. .