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PubMed · 8843150

Constructing an efficient trans-acting genomic HDV ribozyme.

J Kawakami·K Yuda·Y A Suh·P K Kumar·F Nishikawa·H Maeda·K Taira·E Ohtsuka·S Nishikawa
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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).

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Base SequenceDNA PrimersEnzyme ActivationGenetic EngineeringGenome, ViralHepatitis Delta VirusKineticsMolecular Sequence DataMutagenesis, Site-DirectedNucleic Acid ConformationRNA, CatalyticRNA, ViralJournal Article

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J Kawakami, K Yuda, Y A Suh, P K Kumar, F Nishikawa, H Maeda, K Taira, E Ohtsuka, S Nishikawa. 1996-09-30. Constructing an efficient trans-acting genomic HDV ribozyme.. https://doi.org/10.1016/0014-5793(96)00941-6

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