Selection of a high activity ribozyme against cytostatic drug resistance-associated glypican-3 using an in vitro assay containing total tumor RNA.
We tested 11 hammerhead ribozymes for their ability to bind and cleave RNA transcripts of the cytostatic drug resistance-associated glypican-3-encoding gene (GPC3, MXR7, OCI-5). To select the optimum target sequence, the activity of each hammerhead ribozyme was tested in a short in vitro assay using truncated RNA substrates without time-consuming cloning procedures. Glypican-3-derived RNA was cleaved effectively by 3 of 11 hammerhead ribozymes. One of these, the hammerhead ribozyme Rz967, recognized the GUC sequence at nucleotides +965 to +967 of the open reading frame of the glypican-3-encoding mRNA. Rz967 cleaved in vitro-transcribed fragments derived from glypican-3 mRNA (nucleotides +803 to +1036) most efficiently. Cleavage efficiency was confirmed by a rapid in vitro assay using full-length total tumor cell RNA. We were able to demonstrate that this new in vitro assay is suitable for the selection of hammerhead ribozymes that have the capability to cleave glypican-3-encoding mRNA in human tumors.
Wichert
A
A
Institute of Pathology, Humboldt University Berlin, Germany.
Holm
P S
PS
Dietel
M
M
Lage
H
H
D001483:Base Sequence; D002474:Cell-Free System; D018432:Drug Resistance, Multiple; D053673:Glypicans; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011509:Proteoglycans / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D012313:RNA / Q000187:drug effects*; D016337:RNA, Catalytic / Q000627:therapeutic use*; D013997:Time Factors
D053673:Glypicans; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D011509:Proteoglycans; D012313:RNA; D016337:RNA, Catalytic; C121505:Rz967