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Bulletin of the Korean Chemical Society (BKCS)

ISSN 0253-2964(Print)
ISSN 1229-5949(Online)
Volume 27, Number 5
BKCSDE 27(5)
May 20, 2006 

Efficient Target Site Selection for an RNA-cleaving DNAzyme through Combinatorial Library Screening
Ki-Sun Kim, Woo-Hyung Choi, Soo-Jeong Gong, Sangtaek Oh, Jae Hyun Kim, Dong-Eun Kim*
Antisense oligonucleotide, 10-23 DNAzyme, DNAzyme library, Gene inactivation, RNA cleavage
Identification of accessible sites in targeted RNAs is a major limitation to the effectiveness of antisense oligonucleotides. A class of antisense oligodeoxynucleotides, known as the “10-23” DNA enzyme or DNAzyme, which is a small catalytic DNA, has been shown to efficiently cleave target RNA at purinepyrimidine junctions in vitro. We have designed a strategy to identify accessible cleavage sites in the target RNA, which is hepatitis C virus nonstructural gene 3 (HCV NS3) RNA that encodes viral helicase and protease, from a pool of random DNAzyme library. A pool of DNAzymes of 58 nucleotides-length that possess randomized annealing arms, catalytic core sequence, and fixed 5'/3'-end flanking sequences was designed and screened for their ability to cleave the target RNA. The screening procedure, which includes binding of DNAzyme pool to the target RNA under inactive condition, selection and amplification of active DNAzymes, incubation of the selected DNAzymes with the target RNA, and target site identification on sequencing gels, identified 16 potential cleavage sites in the target RNA. Corresponding DNAzymes were constructed for the selected target sites and were tested for RNA-cleavage in terms of kinetics and accessibility. These selected DNAzymes were effective in cleaving the target RNA in the presence of Mg2+. This strategy can be applicable to identify accessible sites in any target RNA for antisense oligonucleotides-based gene inactivation methods.
657 - 662
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