strand (the coding strand) of RNA (commonly mRNA or viral RNA) through formation of an exactly base
paired duplex between the target RNA and an added complementary strand (the antisense strand) (Figure
5.21). Formation of the duplex causes inhibition of expression of a particular gene within cells or in vivo
and the aim is to do this without affecting any other gene.30,31
5.7.1.1 Basic Mechanisms
5.7.1.1.1 Steric Block. This mechanism involves formation of an RNA–DNA duplex to physically
block the RNA and to prevent recognition by a protein or other cellular machinery. For example, binding
of an oligonucleotide close to the 5-cap site^32 (Section 7.2.1) or at the site of initiation of translation in
mRNA^33 (Section 7.3.3) may prevent the ribosome or associated machinery from binding to the RNA and
initiating translation (Figure 5.22). Other RNA processing events that can be interfered with sterically by
duplex formation include nuclear splicing^34 and polyadenylation^35 (Section 7.2.1), both of which are
required for the processing of most mammalian gene transcripts and which involve numerous steps of
RNA–protein recognition. In the case of viral RNA, it is possible to block recognition of essential RNA
binding proteins that are required for virus-specific gene regulation.
5.7.1.1.2 Induction of RNase H. Although steric block activity requires stoichiometric amounts of
complementary added oligonucleotide, a more potent inhibitory effect can often be obtained through
recognition of an RNA–oligonucleotide duplex by the ubiquitous cellular enzyme Ribonuclease H (RNase
H). The normal function of this enzyme is to help the removal of RNA primers in DNA replication
(Section 6.6.3). However, when an RNA sequence is targeted in cells by a complementary oligodeoxynu-
cleotide, RNase H-induced cleavage can occur rapidly, usually close to the centre of the targeted RNA sec-
tion.^36 The loss of intact RNA leads to rapid degradation of the RNA. Thus in the case of mRNA, there is
a concomitant reduction in the level of the encoded protein expressed. Most regions of an mRNA can usu-
ally be targeted by such oligonucleotides, including 3- and 5-untranslated regions.
194 Chapter 5
Figure 5.22 Three alternative mechanisms of steric block action of antisense oligonucleotides acting upon RNA
Figure 5.21 Duplex formed by an antisense oligodeoxyribonucleotide and a target mRNA