Production of Enzymatically Resistant Analogs

Binding of an AS-ODN to its complementary mRNA will inhibit protein expression either by sterically blocking the translation machinery or by promoting recognition by degrading enzymes, such as RNase H (18). The method of antisense-mediated inhibition of protein expression varies depending on where on the target mRNA strand the AS-ODN binds. For example, AS-ODNs that are directed at the 5' region of the target mRNA will form a DNA-RNA complex that blocks protein expression by directly inhibiting the binding of the ribosome to the mRNA. Conversely, AS-ODNs that target regions farther downstream of the 5' terminus can block either elongation or splicing of the mRNA. AS-ODNs can also aid in the destruction of their target mRNA. AS-ODNs containing a stretch of six or more nucleotide bases can form DNA-RNA complexes that are recognized by RNase H (19,20). This enzyme cleaves AS-ODN-mRNA hybrids and thus aids in antisense-mediated decreases in protein expression (21).

Modifications are often introduced into the AS-ODN backbone to increase chemical stability, reduce sensitivity to nuclease digestion, and prevent nonspecific interactions with unintended targets. These modifications allow for the administration of smaller quantities of antisense to achieve efficacy, which reduces the risk of toxicity. Three types of backbone modifications are discussed below along with their chemical properties, that contribute to AS-ODN stability and promote specific interactions with target mRNA.

One of the first modifications developed to reduce AS-ODN sensitivity to nuclease digestion involved replacing a nonbridging oxygen atom at both ends of the AS-ODN chain with a methyl group (see Fig. 2A). The result of this modification was called a methylphosphonate oligodeoxynucleotide (22). This type of modification renders the antisense nuclease resistant. However, because the backbone is now uncharged, this modification reduces antisense activity by decreasing RNase H competency (23).

Another modification developed to increase stability involves replacing a nonbridging oxygen atom at each phosphorus at the two ends of the AS-ODN chain with a sulfur (see Fig. 2A), producing a P-ODN (24). This type of antisense is not completely resistant to nuclease digestion; however, it is still charged, so it is water soluble. P-ODNs have been extensively studied in vitro. These antisense analogs can specifically downregulate many protein targets in cell culture, including protein kinase-a (25,26) intercellular adhesion molecule-1 (27), and bcl-2 (28,29). The introduction of a sulfur atom makes P-ODNs more water soluble, but it also increases nonspecific effects, such as binding to heparin-binding proteins, some of which (e.g., fibronectin, laminin 30) have cell adhesion properties (30). P-ODNs are RNase H competent (31). Activation of this enzyme may help to mediate the antisense effects of P-ODNs.

The PNAs are another form of antisense that were designed to enhance the affinity of binding to target mRNA. PNAs are created by replacing the phosphate deoxyribose backbone with uncharged N-(2-aminoethyl)-glycine linkages (see Fig. 2B). Bases are attached to the glycine amino group via methylene carbonyl linkages (32,33). Because PNAs contain a neutral backbone, this enhances hybridization with target mRNA by decreasing the repulsion between strands (34). However, because PNA-RNA complexes do not act as substrates for RNase H, PNAs must block protein translation sterically to exhibit their antisense effects (35).

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