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-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 Mg(2+) or 3 mM Mn(2+). A metal-specificity switch approach permitted the identification of nonbridging phosphate oxygens (proR(P) or proS(P)) 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 Mg(2+), and this was reversed in the presence of Mn(2+). Further experiments with thio-deoxyribozymes of stereodefined P-chirality suggested direct involvement of both oxygens of the P5 phosphate and the proR(P) oxygen at P9 in the metal ion coordination. In addition, it was found that the oxygen atom at C6 of G(6) contributes to metal ion binding and that this interaction is essential for 10-23 deoxyribozyme catalytic activity.
Nawrot
Barbara
B
Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences, Lodz, Poland. bnawrot@bio.cbmm.lodz.pl
Widera
Kinga
K
Wojcik
Marzena
M
Rebowska
Beata
B
Nowak
Genowefa
G
Stec
Wojciech J
WJ
D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism; D021881:DNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D010755:Organophosphates / Q000737:chemistry* / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013997:Time Factors
D002413:Cations, Divalent; D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; D010755:Organophosphates; C402582:RNA-cleaving DNA 10-23; D008345:Manganese; D008274:Magnesium; D010100:Oxygen