Premium
Constructing an efficient trans ‐acting genomic HDV ribozyme
Author(s) -
Kawakami Junji,
Yuda Kazuhiro,
Suh Young-Ah,
Kumar Penmetcha K.R.,
Nishikawa Fumiko,
Maeda Hidekatsu,
Taira Kazunari,
Ohtsuka Eiko,
Nishikawa Satoshi
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)00941-6
Subject(s) - ribozyme , pseudoknot , vs ribozyme , cleavage (geology) , mammalian cpeb3 ribozyme , cleave , hairpin ribozyme , chemistry , in vitro , stereochemistry , trans acting , nucleotide , biology , rna , microbiology and biotechnology , biochemistry , gene , enzyme , paleontology , fracture (geology) , mutant
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 (k clv = 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 k clv = 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°C with 1 mM of MgCl 2 ).