Nucleic-acid- and cell-based therapeutics - Pharmaceutical Biotechnology: Concepts and Aapplications - page 449

Figure 14.14 Outline of how an antisense oligonucleotide can prevent synthesis of a gene product by
blocking translation. In practice, antisense oligos are 12-18 nucleotides in length. In many instances,
antisense binding is believed to occur in the nucleus
Intramolecular complementary base pairing can occur (particularly within transfer and ribosomal
RNA, but also messenger RNA), resulting in the formation of short duplex sequences, separated
by stems and loops. Such higher-order structure seems to be functionally important, conferring
recognition motifs for proteins and additional nucleic acids, as well as helping to stabilize the
RNA. Regions engaged in intramolecular base pairing are obviously poor targets for antisense oli-
gos. It is thus desirable to synthesize a nucleotide whose sequence is complementary to an acces-
sible sequence along the mRNA backbone. Various approaches are taken to identify such suitable
sequences (remember, the entire sequence of the mRNA will be known). The 'blind' or 'shotgun'
approach entails synthesizing large numbers of oligos targeted to various (often overlapping) re-
gions of the mRNA. The ability of each oligo to block translation of the mRNA is then directly
assessed in an in vitro assay system using cell-free extracts. The second design approach entails
the use of various computer programs to interrogate the mRNA sequence in an attempt to predict
its higher-order structure (and hence identify accessible sequences). This approach remains to be
optimized. The translation initiation sites of mRNAs are often popular targets because they are
essential to translation and they are generally free from secondary structure. However, sequence
homologies can exist within these sequences in unrelated genes. This reduces the specifi city of the
blocking effect and could lead to clinically signifi cant side effects.
Binding results in the blocking of translation of the mRNA and, hence, prevents synthesis of
the mature gene's protein product. The prevention of mRNA translation by duplex formation with
antisense oligonucleotides appears to be underpinned by various mechanisms, including: (a) the
oligonucleotides act as steric blockers, i.e. prevent proteins involved in translation, or other aspects
of mRNA processing, from binding to appropriate sequences in the mRNA; (b) the generation of
duplexes also likely allows targeting by intracellular RNases such as RNaseH. This enzyme is
capable of binding to RNA-DNA duplexes and degrading the RNA portion of the duplex (most
synthetic antisense oligonucleotides are DNA based).