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Figure 2 Antisense mechanisms of action. Cartoon depicting 3 different mechanisms by which an antisense oligonucleotide can inhibit expression of a targeted gene product by hybridization to the mRNA, or pre-mRNA which codes for the gene product.

on a target RNA. In our experience, we have found active oligonucleotides that work through an RNase H-dependent mechanism can hybridize to any region on the mRNA or pre-mRNA. Thus, some serendipity is still involved in the process of identifying and optimizing potent and effective antisense inhibitors.

Early on it was believed that occupancy of the RNA (the receptor for the antisense oligonucleotide) by the oligo-nucleotide would be sufficient to block translation of the RNA (i.e., translation arrest) (34). Subsequent studies have documented that oligonucleotides are not efficient at blocking translation of mRNA if they bind 3' to the AUG translation initiation codon. Furthermore, we have found that only certain sites in the 5'-untranslated region of a mRNA are effective target sites for an antisense oligonucleotide. In particular, the 5'-terminus of a transcript appears to be a good target site for oligonucleotides for some molecular targets in that occupancy of this region prevents assembly of the ribosome on the RNA (35). It should be noted that occupancy of the receptor (RNA) and steric blocking of factor binding by high-affinity oligonucleotides can be an efficient mechanism for blocking gene expression. For the example cited above, the steric blocking oligonucleotide was approximately 10-fold more potent than an oligonucleo-tide that supports RNase H activity. These results suggest that catalytic turnover of the target RNA is not the rate-limiting step for antisense oligonucleotides.

Another process that noncatalytic oligonucleotides can use to alter gene expression is through regulating RNA processing. Most mammalian RNAs undergo multiple post- or cotrans-criptional processing steps, including addition of a 5'-cap structure, splicing, and polyadenylation. Because single-stranded antisense oligonucleotides localize to the cell nucleus (36-39), they have the potential of regulating these processes.

Several studies have been published documenting that antisense oligonucleotides can be used to regulate RNA splicing in both cell-based assays and in rodent tissues (40-47). Oligonucleotides can be used to modulate alternative splicing by promoting use of cryptic splice sites as was exemplified for p thalassemia (40,41), or by enhancing use of an alternative splice site. Oligonucleotide binding to the pre-mRNA can also be exploited to mask polyadenylation signals on the pre-mRNA, forcing the cell to use alternative poly A sites (48). Finally, oligonucleotides, in principle, can regulate RNA function by sterically preventing factors from binding or changing the structure of the RNA such that it is no longer recognized by the factor. Thus, there are multiple mechanisms by which oligonucleotides can be use to inhibit or modulate expression of a target gene product. No single mechanism is far superior to other mechanisms, thus one should tailor the mechanism for the specific biological application.

Figure 3 Positions that have been chemically modified for antisense oligonucleotides.

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