title: Constructing an efficient trans-acting genomic HDV ribozyme.
abstract: We have engineered a genomic HDV ribozyme to construct several trans-acting ribozymes for use in trans to cleave target RNAs. Among the 10 different combinations attempted, only HDV88-Trans had cleavage activity on the 13-nucleotide substrate, R13, in vitro. To improve the cleavage efficiency, at least in vitro, of the HDV88-Trans ribozyme (kclv = 0.022 min(-1)), we have constructed several variants that differ in forming stem II (length) in the pseudoknot secondary structure model. When cleavage rate constants were analyzed and compared among variants of HDV88-Trans, HDV88-Trans-4 yielded kclv = 1.7 min(-1). HDV88-Trans-4 thus represents the highest active genomic HDV ribozyme that functions in trans thus far constructed, and has activity under physiological conditions (pH 7.1 at 37 degrees C with 1 mM of MgCl2).
authors.affiliation: Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Kawakami
authors.forename: K
authors.initials: K
authors.lastname: Yuda
authors.forename: Y A
authors.initials: YA
authors.lastname: Suh
authors.forename: P K
authors.initials: PK
authors.lastname: Kumar
authors.forename: F
authors.initials: F
authors.lastname: Nishikawa
authors.forename: H
authors.initials: H
authors.lastname: Maeda
authors.forename: K
authors.initials: K
authors.lastname: Taira
authors.forename: E
authors.initials: E
authors.lastname: Ohtsuka
authors.forename: S
authors.initials: S
authors.lastname: Nishikawa
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D004789:Enzyme Activation; D005818:Genetic Engineering; D016679:Genome, Viral; D003698:Hepatitis Delta Virus / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D016337:RNA, Catalytic; D012367:RNA, Viral