Development of 8–17 XNAzymes that are functional in cells

DNA enzymes (DNAzymes), which cleave target RNA with high specificity, have been widely investigated as potential oligonucleotide-based therapeutics. Recently, xeno-nucleic acid (XNA)-modified DNAzymes (XNAzymes), exhibiting cleavage activity in cultured cells, have been developed. However, a versatile approach to modify XNAzymes that function in cells has not yet been established. Here, we report an X-ray crystal structure-based approach to modify 8–17 DNAzymes; this approach enables us to effectively locate suitable XNAs to modify. Our approach, combined with a modification strategy used in designing antisense oligonucleotides, rationally designed 8–17 XNAzyme (“X8–17”) that achieved high potency in terms of RNA cleavage and biostability against nucleases. X8–17, modified with 2′-O-methyl RNA, locked nucleic acid and phosphorothioate, successfully induced endogenous MALAT-1 and SRB1 RNA knockdown in cells. This approach may help in developing XNAzyme-based novel therapeutic agents.