Identification of phosphate oxygens that are important for self-cleavage activity of the HDV ribozyme by phosphorothioate substitution interference analysis.
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 self-cleavage 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.
Jeoung
Y H
YH
National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, MITI, Ibaraki, Japan.
Kumar
P K
PK
Suh
Y A
YA
Taira
K
K
Nishikawa
S
S
D001483:Base Sequence; D003698:Hepatitis Delta Virus / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010100:Oxygen / Q000502:physiology*; D010710:Phosphates; D012322:RNA Precursors / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D013873:Thionucleotides
D010710:Phosphates; D012322:RNA Precursors; D016337:RNA, Catalytic; D012367:RNA, Viral; D013873:Thionucleotides; D010100:Oxygen