Correlation of activity with stability of chemically modified ribozymes in nuclei suspension.
To examine hammerhead ribozyme activity in the nuclear environment, we have used nuclei isolated from HTLV-I tax transformed fibroblasts to evaluate ribozymes targeted against HTLV-I tax RNA. The ribozyme activity in nuclei suspension was strongly dependent on the resistance of the particular ribozyme to endogenous nucleases. A ribozyme containing exclusively 2'-deoxynucleotides in its stems cleaved target RNA by its catalytic activity in the absence of proteins and caused degradation in their presence by induction of nuclear RNase H activity. A ribozyme containing 2'-amino- and 2'-fluoropyrimidine nucleosides in combination with terminal phosphorothioate linkages was significantly more stable in nuclei suspension and also exhibited a more than threefold higher cleavage efficacy than its unmodified counterpart. The increased resistance against nuclease degradation is mainly due to terminal phosphorothioate linkages, suggesting that both 5' and 3'-exonucleases are primarily responsible for the nuclear degradation of oligonucleotides.
Heidenreich
O
O
Scripps Research Institute, Department of Molecular and Experimental Medicine, La Jolla, CA 92037, USA.
Xu
X
X
Swiderski
P
P
Rossi
J J
JJ
Nerenberg
M
M
D000818:Animals; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism*; D002472:Cell Transformation, Viral; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D016356:Gene Products, tax / Q000235:genetics* / Q000378:metabolism; D015368:Human T-lymphotropic virus 1; D051379:Mice; D008822:Mice, Transgenic; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D016356:Gene Products, tax; D016337:RNA, Catalytic; D012367:RNA, Viral