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

Abstract

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.

Hydrolysis of Bulged Nucleotides in Hybrids Formed by Rna and Imidazole-Derivatized Oligo-2′-O-Methylribonucleotides

“…These molecules have been shown to cleave target RNAs at substoichiometric concentrations, at considerably faster rates than reported in this study [19,20] , a result that suggests the possibility of catalytic turnover. Although the antisense molecules reported in this study possess a rather modest rate of hydrolysis, further modifications of the tether or functional groups may result in greater hydrolytic efficiency and consequently provide antisense oligonucleotides that could be used to attack cellular targets.…”

STUDIES IN OLIGONUCLEOTIDE-BASED ARTIFICIAL NUCLEASE SYSTEMS. INTRAMOLECULAR COPPER (II) COMPLEX FORMATION IN AN OLIGONUCLEOTIDE BIS-PHENANTHROLINE CONJUGATE

“…[1,2] Our OBANs hybridize giving bulge formation in the central region of the RNA target. [3] In search of optimal combination of linker and linker attachment position we have prepared novel 2'-O-Me-oligoribonucleotide conjugates ( IV--VI) carrying dmp moieties.…”

Zinc Ion-Dependent Peptide Nucleic Acid-Based Artificial Enzyme that Cleaves RNA—Bulge Size and Sequence Dependence

“…We have designed artificial nucleases based on the concept that the modified oligonucleotide part of the artificial nuclease recognizes the target RNA upon hybridization and forces the RNA to form a bulge. This concept enabled the development of artificial enzymes capable of cleaving target RNA sequences with turnover [ 20 , 21 , 22 ]. Previous efforts have also resulted in the development of peptide nucleic acid-based artificial nucleases (PNAzymes) [ 23 , 24 , 25 , 26 ].…”