Constructing an efficient trans-acting genomic HDV ribozyme.
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).
Kawakami
J
J
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Yuda
K
K
Suh
Y A
YA
Kumar
P K
PK
Nishikawa
F
F
Maeda
H
H
Taira
K
K
Ohtsuka
E
E
Nishikawa
S
S
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*
D017931:DNA Primers; D016337:RNA, Catalytic; D012367:RNA, Viral