Nuclease-resistant chimeric ribozymes containing deoxyribonucleotides and phosphorothioate linkages.
Hammerhead ribozymes are considered to be potential therapeutic agents for HIV virus because of their site-specific RNA cleavage activities. In order to elucidate structure--function relationship and also to hopefully endow ribozymes with resistance to ribonucleases, we firstly synthesized chimeric DNA/RNA ribozymes in which deoxyribonucleotides were substituted for ribonucleotides at noncatalytic residues (stems I, II, and III). Kinetic analysis revealed that (i) DNA in the hybridizing arms (stems I and III) enhanced the chemical cleavage step. (ii) stem II and its loop do not affect its enzymatic activity. Secondly, we introduced deoxyribonucleotides with phosphorothioate linkages to the same regions (stems I, II, and III) in order to test whether such thio-linkages further improve their resistance to nucleases. Kinetic measurements revealed that this chimeric thio-DNA/RNA ribozyme had seven-fold higher cleavage activity (kcat = 27 min-1) than that of the all-RNA ribozyme. In terms of stability in serum, DNA-armed ribozymes gained about 10-fold higher stability in human serum but no increase in stability was recognized in bovine serum, probably because the latter serum mainly contained endoribonucleases that attacked unmodified catalytic-loop regions of these ribozymes. Thirdly, in order to protect them from endoribonucleases, three additional modifications were made at positions U7, U4 and C3 within the internal catalytic-loop region, that succeeded in gaining more than a hundred times greater resistance to nucleases in both serums. More importantly, these catalytic-loop modified ribozymes had the comparable cleavage activity (kcat) to the wild-type ribozyme. Since these chimeric thio-DNA/RNA ribozymes are more resistant to attack by both exonucleases and endoribonucleases than the wild-type all-RNA ribozymes in vivo and since their cleavage activities are not sacrificed, they appear to be better candidates than the wild type for antiviral therapeutic agents.
Shimayama
T
T
National Institute of Bioscience and Human Technology, Agency of Industrial Science & Technology, MITI, Tsukuba Science City, Japan.
Nishikawa
F
F
Nishikawa
S
S
Taira
K
K
D000818:Animals; D001483:Base Sequence; D001769:Blood; D002417:Cattle; D002678:Chimera; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D003854:Deoxyribonucleotides; D004355:Drug Stability; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism; D013873:Thionucleotides; D013997:Time Factors
D003854:Deoxyribonucleotides; D016337:RNA, Catalytic; D013873:Thionucleotides; D004247:DNA; D003851:Deoxyribonucleases; D012260:Ribonucleases