Oligonucleotide based artificial nuclease (OBAN) systems. Bulge size dependence and positioning of catalytic group in cleavage of RNA-bulges

Three zinc ion dependent oligonucleotide based artificial nucleases (OBANs) have been synthesized. These consist of 2'-O-methyloligoribonucleosides connected to 5-amino-2,9-dimethylphenanthroline via a urea function to a linker extending either from C-5 of deoxyuridine or from the 2'-position of uridine moieties. Both types of linkers are placed centrally in the modified sequence and in addition one OBAN carries the C-5 modified dU as an additional nucleoside unit at the 5'-end. All three OBANs are shown to cleave target oligoribonucleotides selectively. The target RNA's are varied to form differently sized bulges (0-5 nucleotides (nt)) and the different OBANs have different preferences for which sizes are preferentially cleaved. The OBAN with the centrally positioned C-5 linked zinc chelate preferentially cleaves 3 and 4-nt bulges, the OBAN with the 2'-linked chelate has a preference for slightly smaller bulges and the OBAN with a 5'-end chelate is more efficient the larger the bulge is. In addition the OBAN with the centrally positioned C-5 linked zinc chelate is shown to be a real enzyme, capable of turnover of substrate and displaying Michaelis-Menten behaviour. The main differences in efficiency of cleavage between the different OBAN-RNA substrate combinations are likely to be due to proximity factors i.e. the positioning of a catalytic group relative to cleaved phosphodiester functions. The model systems investigated partially display the importance of catalytic group positioning and should be useful in future development of more efficient OBANs.