In vitro evolution of the hammerhead ribozyme to a purine-specific ribozyme using mutagenic PCR with two nucleotide analogues

The conventional hammerhead ribozyme cleaves RNA 3' to nucleotide triplets with the general formula NUH, where N is any nucleotide, U is uridine and H is any nucleotide except guanosine. In order to isolate hammerhead ribozym e sequences capable of cleaving 3' to the GUG triplet, we performed a mutag enic selection protocol starting with the conventional sequence of an NUH-c leaving ribozyme. The 22 nucleotides in the core and the stem-loop II regio n were subjected to mutagenic PCR using the two nucleotide analogues 6-(2-d eoxy-beta-D-ribofuranosyl)-3,4dihydro-8H pyrimido-[4,5-C)][1,2] oxazin-7-on e and of 8-oxo-2'-deoxyguanosine. After five repetitions of the selection c ycle, several clones showed cleavage activity. One sequence, having one del etion, showed at least a 90 times higher in trans cleavage rate than the st arting ribozyme. It cleaved 3' to GUG and GUA. The sequence of this ribozym e is essentially identical with that obtained previously by selection for A UG cleavage starting with a randomised core and stem-loop II region. This i dentical result of two independent selection procedures supports the notion that sequences for NUR cleavage, where R is a purine nucleotide, are not c ompatible with the classical hammerhead structure, and that the sequence sp ace for this cleavage specificity is very limited. The cleavage of NUR trip lets is not restricted to the sequence of the substrate that was used for s election but is sequence-independent for in trans cleavage, although the se quence context influences the value for the cleavage rate somewhat. Analysi s of cleavage activities indicates the importance of A at position L2.5 in loop II. (C) 2000 Academic Press.