How to Choose Optimal Antisense Targets in an mRNA
One of the major challenges of an antisense experiment is the identification of sequences within the target RNA, which are accessible by an antisense oligonucleotide. Three-dimensional folding of the RNA results in both inaccessible segments in the inner part of the RNA molecule and double stranded regions. The folding of RNAs may prevent many oligonucleotides from reaching their complementary regions (Branch, 1998). While it is impossible to predict accessible target sites for antisense oligonucleotides from computer-aided models of RNA-structure (Fig.2), combinatorial screening is a promising approach to search for such sequences. The identification of the most active antisense oligonucleotide however remains empirical and has to be performed in translation arrest assays and in cell culture. Sequences with well-known non-antisense effects have to be avoided. Finally, stringent controls are required to prove an antisense-mediated mechanism of action.
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