Detection of antisense and ribozyme accessible sites on native mRNAs: Application to NCOA3 mRNA

The efficacies of antisense oligonucleotides and ribozymes are greatly depe ndent on the accessibility of their mRNA targets. Target site accessibility is affected by both RNA structure and the proteins associated along the le ngth of the RNA. To mimic the native state of mRNA for site identification, we have previously used endogenous mRNAs in cellular extracts as targets f or defined sequence oligodeoxynucleotides (ODNs) designed to identify both antisense pairing and potential ribozyme cleavage sites. The rationale for this approach is that the specific pairing of an ODN with a mRNA forms a DN A:RNA hybrid that is cleaved by the endogenous RNaseH in the cell extract. To extend the usefulness of this basic approach, we report here the use of semi-random ODN libraries to identify hammerhead ribozyme cleavage sites. T hus, the most accessible sites for antisense and ribozyme base pairing are selected by this approach. A novel feature of the approach described here i s the use of terminal transferase-dependent PCR (TDPCR) after reverse trans cription to estimate the cleavage efficiency and to precisely determine the RNaseH and ribozyme cleavage sites on mRNAs in cell extracts following tre atment with ODN or ribozyme libraries. As a model system, we have targeted the NCOA3 (also known as AIB-1) mRNA in cell extracts. The NCOA3 mRNA encod es a nuclear receptor co-activator that is amplified and overexpressed in a high proportion of breast and ovarian cancers. A highly accessible site on this mRNA was identified, and a ribozyme targeted to this site was demonst rated to effectively downregulate NCOA3 function in cells.