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ACS Comb Sci. 2013 Apr 8;15(4):174-82. doi: 10.1021/co3001378. Epub 2013 Mar 13.

Toward the combinatorial selection of chemically modified DNAzyme RNase A mimics active against all-RNA substrates.

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1
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver BC, V6T 1Z1, Canada.

Abstract

The convenient use of SELEX and related combinatorial methods of in vitro selection provides a formidable gateway for the generation of DNA enzymes, especially in the context of improving their potential as gene therapeutic agents. Here, we report on the selection of DNAzyme 12-91, a modified nucleic acid catalyst adorned with imidazole, ammonium, and guanidinium groups that provide for efficient M(2+)-independent cleavage of an all-RNA target sequence (kobs = 0.06 min(-1)). While Dz12-91 was selected for intramolecular cleavage of an all-RNA target, it surprisingly cleaves a target containing a lone ribocytosine unit with even greater efficiency (kobs = 0.27 min(-1)) than Dz9-86 (kobs = 0.13 min(-1)). The sequence composition of Dz12-91 bears a marked resemblance to that of Dz9-86 (kobs = 0.0014 min(-1) with an all-RNA substrate) that was selected from the same library to cleave a target containing a single ribonucleotide. However, small alterations in the sequence composition have a profound impact on the substrate preference and catalytic properties. Indeed, Dz12-91 displays the highest known rate enhancement for the M(2+)-independent cleavage of all-RNA targets. Hence, Dz12-91 represents a step toward the generation of potentially therapeutically active DNAzymes and further underscores the usefulness of modified triphosphates in selection experiments.

PMID:
23485334
DOI:
10.1021/co3001378
[Indexed for MEDLINE]

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