Synthesis and enzymic hydrolysis of oligoribonucleotides incorporating 3-deazaguanosine: The importance of the nitrogen-3 atom of single conserved guanosine residues on the catalytic activity of the hammerhead ribozyme

Abstract

Four base-modified hammerhead ribozyme/substrate complexes were constructed in which single guanosine (1) residues were replaced by 3-deazaguanosine (2) in the positions G(5), G(8), G(L2.1), and G(12). The base-modified ribozyme complexes were prepared by solid-phase synthesis of oligoribonucleotides employing the novel phosphoramidite 3 derived from 2. Phosphoramidite 3 carried a phenoxyacetyl group at the amino function and a diphenylcarbamoyl residue at the oxo group of the nucleobase. The 2'-hydroxy group was blocked with a triisopropylsilyl residue. Kinetic analysis of the phosphodiester hydrolysis showed a moderate decrease of the ribozyme catalytic activity when the residues G5 or G8 were replaced by 3-deazaguanosine and a 200-fold decrease when G(12) was substituted. A 6-fold catalytic increase occurred when 3-deazaguanosine was replacing G(L2.1) in the loop region. The data indicate that the N(3) atom of compound 2, in particular at position G(12) is critical for the ribozyme activity.