title: Inhibition of hepatitis B virus X gene expression by novel DNA enzymes.
abstract: 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 Mg(2+)-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.
authors.affiliation: Laboratory of Virology, National Institute of Immunology, JNU Campus, Aruna Asaf Ali Marg, New Delhi-110067, India.
authors.forename: R
authors.initials: R
authors.lastname: Goila
authors.forename: A C
authors.initials: AC
authors.lastname: Banerjea
mesh_terms: D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D017931:DNA Primers; D003851:Deoxyribonucleases / Q000378:metabolism*; D015536:Down-Regulation; D015971:Gene Expression Regulation, Enzymologic; D016325:HIV Long Terminal Repeat; D006801:Humans; D007700:Kinetics; D011401:Promoter Regions, Genetic; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D017931:DNA Primers; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D003851:Deoxyribonucleases