Exon skipping is one of a number of ‘‘steric blocking’’applications of oligonucleotides (ONs) and their analogues that in recent years have undergone a renaissance and which have led to new therapeutic opportunities (Kurreck, 2008; Kole et al., 2012; Lightfoot and Hall, 2012). Whereas most standard antisense applications involve cleavage of the RNA by intracellular ribonuclease H (RNase H) or by argonaute2 (Ago2) enzymes, exon skipping by steric blocking merely requires binding of the ON in an antisense orientation to an RNA target and blockage of the important biological function of splicing. In exon skipping, as well as the similar splice-switching activity of exon inclusion, the target is premRNA located in the cell nucleus. Thus, the ON must enter the cell nucleus in sufficient quantity to be in excess over the target pre-mRNA, bind to it strongly, and interfere with the splicing machinery. For drug use, the ON must exhibit additionally a number of other important properties such as good biodistribution to the target organ (s), lack of immune recognition effects, and a good therapeutic window between effective and toxic doses. Further, ON synthesis must be routinely achievable on varying scales. Such simultaneous requirements have taxed the ingenuity of chemists in ON design.

It is necessary in all therapeutic applications of ONs to include modifications to the ON backbone and/or sugar component to protect against nuclease degradation. Interestingly, the very first analogue to find use therapeutically was the phosphorothioate linkage (Matsukura et al., 1987), which is still utilized in most ONs in current clinical trials. Further modifications have followed in subsequent years that have improved stability to nucleases, or even result in the ON being nuclease inert. The best ON analogues both enhance RNA binding strength, compared with an unmodified ON, and reduce nuclease resistance. Increased binding strength has allowed shorter ONs to be used in some cases, thus reducing the chance of binding to an incorrect RNA site through partial sequence complementarity. For example, in the case of ONs containing locked nucleic acids (LNAs), lengths of 12 to 15 are usually used (Lanford et al., 2010; Straarup et al., 2010) and even shorter in the case of all-LNA ONs (Obad et al., 2011). More commonly, lengths of ON analogue synthesized for exon skipping and other steric blocking applications are 14–30 residues.