adjuvants. Immune stimulation of this or similar type has also been used to explain in vivoactivities of
some supposed antisense oligonucleotides that contain CpG sequences.
5.7.1.4.2 DNA Aptamers. Oligodeoxynucleotides have been selected (Section 5.7.3) that bind spe-
cific cellular proteins. Such aptamerscan fold into unusual structures (such as a G quadruplex, Section
2.3.7). A chemically synthesised DNA aptamer that binds strongly to vascular endothelial growth factor
has been formulated as a conjugate with polyethylene glycol and has recently been given regulatory
approval (Macugen) for treatment of patients with the wet form of age-related macular degeneration, a
common cause of blindness due to abnormal blood vessel growth.^41
5.7.2 RNA Interference
In the late 1990s, gene silencing by double-stranded RNA was observed in plants in the laboratory of
David Baulcombe^42 and in the worm C. elegansin that of Craig Mello.^43 Within a very few years gene
silencing activities were found in many diverse organisms and have gone on to be harnessed as powerful
diagnostic reagents in genome research and as potential therapeutics.44–46RNA interference(RNAi) prob-
ably evolved from the need for eukaryotic cellular defence against foreign (e.g.viral) duplex RNA or DNA
that is transcribed into RNA. Distinct but overlapping pathways have been elucidated that represent dif-
ferent forms of genetic regulation (Figure 5.24).
Primary RNA transcripts in the nucleus has been found to contain hundreds of endogenous sequences
known as microRNAs(miRNAs) that can fold into hairpins that contain imperfect matches (Section 7.5.3).
The transcripts are processed by the nuclear complex Drosha, which contains an RNase III activity that
cleaves such RNAs to produce hairpins of about 70 residues (pre-microRNAs). After export to the cytosol, the
Nucleic Acids in Biotechnology 197
Figure 5.24 Mechanisms of action involved in RNA interference (RNAi). Pathway (a) shows steps in the processing
of microRNA (miRNA) eventually leading to inhibition of translation. Pathway (b) shows the processing
of double-stranded viral RNA to form short interfering RNA (siRNA) and eventual cleavage of mRNA
by the RNA-induced silencing complex (RISC) complex. ShortRNA (shRNA) (centre) can in principle
enter either pathway