High activity and stability of hammerhead ribozymes containing 2'-modified pyrimidine nucleosides and phosphorothioates.
The influence of chemical modifications on the catalytic activity and stability of a hammerhead ribozyme directed against the long terminal repeat RNA of the human immunodeficiency virus 1 was examined. Previous studies had shown that substitution of all pyrimidine nucleosides by their 2'-fluoro analogs led to an 8-fold decrease in catalytic efficiency in the cleavage reaction compared to the unmodified ribozyme (Heidenreich, O., and Eckstein, F. (1992) J. Biol. Chem. 267, 1904-1909). It is shown here that replacement of the 2'-fluoro-2'-deoxyuridines in the conserved region of this ribozyme, positions 4 and 7, by 2'-amino-2'-deoxyuridines fully restores catalytic activity of the ribozyme. Ribozymes containing these 2'-modifications show an increased stability against RNases present in fetal calf serum and in cell culture supernatant. The stability is increased further by the incorporation of four terminal phosphorothioates as protection against 3'-exonucleases, the degree of which depends on the secondary structure of the ribozyme. Such ribozymes are stable in undiluted fetal calf serum for at least 24 h. The results clearly demonstrate the potential to design stable ribozymes without any loss of catalytic activity.
Heidenreich
O
O
Max-Planck-Institut für Experimentelle Medizin, Göttingen, Federal Republic of Germany.
Benseler
F
F
Fahrenholz
A
A
Eckstein
F
F
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004795:Enzyme Stability; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011741:Pyrimidine Nucleosides / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D012260:Ribonucleases / Q000097:blood; D013873:Thionucleotides / Q000378:metabolism*
D011741:Pyrimidine Nucleosides; D016337:RNA, Catalytic; D013873:Thionucleotides; D012260:Ribonucleases