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Nucleic Acids Research, 1994, Vol. 22, No. 18 3722-3727
© 1994

RNA

Identification of phosphate oxygens that are important for self-cleavage activity of the HDV ribozyme by phosphorothioate substitution interference analysis

Yeon-Hee Jeoung$, P.K.R. Kumar, Young-Ah Suh§, Kazunari Taira and Satoshi Nishikawa*

National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology MITI, Tsukuba Science City, Ibaraki 305, Japan

*To whom correspondence should be addressed

Received June 6, 1994. Revised August 2, 1994. Accepted August 2, 1994.

A phosphorothioate substitution interference assay was used to investigate the role of the pro-Rp oxygens of phosphate groups in the self-cleavage reaction of the genomic human hepatitis delta virus (HDV) ribozyme. Incorporation of several different phosphorothioates (NTP{alpha}S) into the HDV ribozyme inhibited the self-cleavage activity. Incorporation of uridine 5' phosphorothioate or adenosine 5' phosphorothioate maintained 72% of the original selfcleavage activity whereas incorporation of guanosine 5' phosphorothioate or cytosine 5' phosphorothioate into the precursor reduced self-cleavage activity to about 20% in each case. Using partially substituted phosphorothioate-modified transcripts, we identified the pro-Rp oxygens that are important for the ribozyme activity, and they are located at positions 0,1,4, 5, 21, 24, 25, 27, 28, 30–34, 40, 43 and 75. In particular, the pro-Rp oxygens at positions 0, 1 and 21 are appear to be critical for the self-cleavage activity of the HDV ribozyme.

$ Present addresses: Department of Chemistry, Korea Advanced Institute of Science and Technology, Taejon

§ Present addresses: Department of Life Science, Pohang University of Science and Technology, Pchang, Korea


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