The three guanosines of the central core of a hammerhead ribozyme were replaced by 2-aminopurine ribonucleoside, xanthosine, isoguanosine, inosine, and deoxyguanosine. These analogues were incorporated by automated solid-phase synthesis, with the exception of isoguanosine. This was introduced by ligating a donor, which carried the isoguanosine at its 5'-end, and an acceptor oligoribonucleotide by a T4 DNA ligase-catalyzed reaction. Most of these modifications lowered the rate constant of cleavage by the hammerhead ribozyme drastically. Inspection of the possible hydrogen-bonding interactions disturbed by these modifications suggests that there is no G12A9 or A13G8 mismatched base pair in the central region. Increasing the Mg2+ concentration from 10 to 50 mM did not enhance these rates appreciably. This makes it improbable that the guanosines, including their 2'-hydroxyl groups, are involved in the binding of the catalytically active Mg2+. Transition-state destabilizing energies of 0.6-4.7 kcal mol-1 suggest that essentially all guanosines are involved in a hydrogen-bonding network.
Isoguanosine has been incorporated into a 34-mer hammerhead ribozyme by the solid-phase phosphoramidite method, using an acetamidine base protecting group. The activity of the hammerhead ribozyme when singly mutated to isoguanosine at the adenosine positions 6, 9, and 13 was 1-2-fold less than the wild-type activity. Mutations to 2-aminopurine ribonucleoside at positions 9 and 13 were 5-fold reduced in activity, but that at position 6 was approximately 30-fold reduced. These results support the view that the 6-amino functions of A6, A9, and A13 are not very important for catalysis. The 2-position of A6 tolerates a carbonyl function but not an amino group, whereas A9 and A13 tolerate both functional groups. The tolerance of a 2-amino group at A9 and A13 makes G(anti)/A(anti) Watson-Crick type base mispairing for G12/A9 and A13/G8 unlikely.
Seven new steroidal polyamines derived from bile acids, either lithocholic or deoxycholic acid, have been studied as DNA-binding agents using four complimentary methods: an ethidium displacement assay, observed changes in the thermal denaturation of poly[d(AT)], effects on hyperchromicity of DNA, and circular dichroism. In addition, modelling studies were conducted to examine the electrostatic surface potential of the polycations. The results point to a key role for a large hydrophobic surface area on the steroid in addition to the Coulombic attraction by ammonium and guanidinium groups on the steroid interacting with the polyphosphate backbone.
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