Protective effects of anti-ricin A-chain RNA aptamer against ricin toxicity.
OBJECTIVE
To investigate the therapeutic potential of an RNA ligand (aptamer) specific for the catalytic ricin A-chain (RTA), the protective effects of a 31-nucleotide RNA aptamer (31RA), which formed a high affinity complex with RTA, against ricin-induced toxicity in cell-based luciferase translation and cell cytotoxicity assays were evaluated.


METHODS
To test the therapeutic potential of anti-RTA aptamers in Chinese hamster ovary (CHO) AA8 cells stably transfected with a tetracycline regulatable promoter, ricin ribotoxicity was measured using luciferase and ricin-induced cytotoxicity was ascertained by MTS cell proliferation assay with tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium].


RESULTS
Inhibition of protein synthesis by ricin in CHO AA8 cells resulted in diminished luciferase activity and treatment with polyclonal antibody against deglycosylated RTA (dgA) neutralized the inhibitory effects of ricin on luciferase activity and protected against ricin-induced cytotoxicity as measured by MTS assay. The 31RA anti-RTA aptamer inhibited the translation of luciferase mRNA in cell-free reticulocyte translation assay. 31RA aptamer also partially neutralized the inhibitory effects of ricin on luciferase activity and partially protected against ricin-induced cytotoxicity in CHO AA8 cells.


CONCLUSIONS
We have shown that anti-RTA RNA aptamer can protect against ricin ribotoxicity in cell-based luciferase and cell cytotoxicity assays. Hence, RNA aptamer that inhibits RTA enzymatic activity represents a novel class of nucleic acid inhibitor that has the potential to be developed as a therapeutic agent for the treatment of ricin intoxication.
Fan
Shaoan
S
Department of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, New York 10987, United States.
Wu
Feng
F
Martiniuk
Frank
F
Hale
Martha L
ML
Ellington
Andrew D
AD
Tchou-Wong
Kam-Meng
KM
D000818:Animals; D000931:Antidotes / Q000378:metabolism / Q000494:pharmacology*; D052157:Aptamers, Nucleotide / Q000378:metabolism / Q000494:pharmacology*; D016466:CHO Cells; D049109:Cell Proliferation / Q000187:drug effects*; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D007700:Kinetics; D008156:Luciferases / Q000235:genetics; D014176:Protein Biosynthesis / Q000187:drug effects*; D011500:Protein Synthesis Inhibitors / Q000378:metabolism / Q000633:toxicity*; D012276:Ricin / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D014162:Transfection
D000931:Antidotes; D052157:Aptamers, Nucleotide; D011500:Protein Synthesis Inhibitors; D012276:Ricin; D008156:Luciferases