A METHOD FOR SCREENING ANTISENSE OLIGODEOXYRIBONUCLEOTIDES EFFECTIVE FOR MESSENGER-RNA TRANSLATION-ARREST

A transcription and translation coupled reticulocyte lysate system was established for rapid screening of antisense oligodeoxyribonucleotide s (ODNs) to determine which are most effective for mRNA translation-ar rest. A plasmid containing the target cDNA under the control of the T7 (or SP6) promoter was added to the lysate system in the presence of t he T7 (or SP6) RNA polymerase, RNase H, and the antisense ODN under te st. Transcription and translation were accomplished in a one-tube reac tion. Translation-arrest caused by antisense ODN was evaluated in term s of the amounts of de novo-synthesized, [S-35]-methionine or [S-35]cy steine labeled target protein measured by gel electrophoresis and auto radiography. The properties of this system and optimal reaction condit ions for use in antisense ODN screening were determined. Our method is simpler and more rapid than other in vitro screening methods.