Two mono- and a di-RNA-cleaving DNA enzymes with the 10–23 catalytic motif were synthesized that were targeted to cleave at the conserved site/sites of the X gene of the hepatitis B virus. In each case, protein-independent but Mg2+-dependent cleavage of in vitro-synthesized full-length X RNA was obtained. Specific cleavage products were obtained with two different mono- and a di-DNA enzyme, with the latter giving rise to multiple RNA fragments that retained the cleavage specificity of the mono-DNA enzymes. A relatively less efficient cleavage was also obtained under simulated physiological conditions by the two mono-DNA enzymes but the efficiency of the di-DNA enzyme was significantly reduced. A single nucleotide change (G to C) in the 10–23 catalytic motif of the DNA enzyme 307 abolished its ability to cleave target RNA completely. Both, mono- and di-DNA enzymes, when introduced into a mammalian cell, showed specific inhibition of X-gene-mediated transactivation of reporter-gene expression. This decrease was due to the ability of these DNA enzymes to cleave X RNA intracellularly, which was also reflected by significant reduction in the levels of X protein in a liver-specific cell line, HepG2. Ribonuclease protection assay confirmed the specific reduction of X RNA in DNA-enzyme-treated cells. Potential in vivo applications of mono- and di-DNA enzymes in interfering specifically with the X-gene-mediated pathology are discussed.

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