title: Introduction of guanidinium-modified deoxyuridine into the substrate binding regions of DNAzyme 10-23 to enhance target affinity: implications for DNAzyme design.
abstract: Deoxyribozymes (DNAzymes) are important catalysts for potential therapeutic RNA destruction and no DNAzyme has received as much notoriety in terms of therapeutic use as the Mg(2+)-dependent RNA-cleaving DNAzyme 10-23 (Dz10-23). As such, we have investigated the synthetic modification of Dz10-23 with a guanidinium group, a functionality that reduces the anionic nature and can potentially enhance the membrane permeability of oligonucleotides. To accomplish this, we synthesized a heretofore unknown phosphoramidite, 5-(N,N'-biscyanoethoxycarbonyl)-guanidinoallyl-2'-deoxyuridine and then incorporated it into oligonucleotides via solid phase synthesis to study duplex stability and its effect on Dz10-23. This particular modification was chosen as it had been used in the selection of Mg(2+)-free self-cleaving DNAzymes; as such this will enable the eventual comparison of modified DNAzymes that do or do not depend on Mg(2+) for catalysis. Consistent with antecedent studies that have incorporated guanidinium groups into DNA oligonucleotides, this guanidinium-modified deoxyuridine enhanced the thermal stability of resulting duplexes. Surprisingly however, Dz10-23, when synthesized with modified residues in the substrate binding regions, was found to be somewhat less active than its non-modified counterpart. This work suggests that this particular system exhibits uniform binding with respect to ground state and transition state and provides insight into the challenge of re-engineering a Mg(2+)-dependent DNAzyme with enhanced catalytic activity.
authors.affiliation: Chemistry Department, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Curtis H
authors.initials: CH
authors.lastname: Lam
authors.forename: David M
authors.initials: DM
authors.lastname: Perrin
mesh_terms: D055162:Biocatalysis; D021881:DNA, Catalytic / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D003857:Deoxyuridine / Q000378:metabolism*; D015195:Drug Design; D019791:Guanidine / Q000378:metabolism*; D007700:Kinetics; D013379:Substrate Specificity
chemical_list: D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; C402582:RNA-cleaving DNA 10-23; D019791:Guanidine; D003857:Deoxyuridine