Chemistry Reference
In-Depth Information
of which also aim to inhibit the expression of a particular gene.
6
Synthetic ribozymes
mimic the mode of action of naturally occurring ribozymes,
7
which bind to mRNA
and cleave a precise phosphodiester bond. One of the most commonly studied is
the hammerhead ribozyme, which contains two binding arms for substrate recogni-
tion and a catalytic domain for cleavage.
8
Like AS-O, ribozymes have been chemi-
cally modifi ed to increase their stability in cells and their delivery to specifi c tissues
without compromising activity, and some of them are currently in clinical trials for
evaluation as therapeutic agents.
9
The recent discovery of RNA interference (RNAi)
10
has triggered the use of
technologies based on this natural gene-silencing mechanism in biological research
and their evaluation for the treatment of many diseases.
11,12
RNAi is mediated by
small interfering RNAs (siRNAs), which are double-stranded oligoribonucleotides
typically 21-25 nucleotides in length, and two nucleotide 3
- OH overhangs that are
incorporated into an active ribonucleoprotein complex, the RNA-induced silencing
complex (RISC).
13
Unwinding of the two chains of the siRNA duplex is followed
by hybridization of the antisense strand with mRNA, which, depending on the
degree of complementarity, either guides RNA cleavage by the activated RISC
complex or blocks translation. Synthetic siRNAs seem to be more active than AS-O
and could very possibly be developed as a new therapeutic strategy. However, non-
specifi c effects and toxicity may be associated with high activity, and, although
siRNAs are fairly stable in cells, chemical modifi cations that increase cellular stabil-
ity and facilitate delivery may be required to ensure their effi ciency
in vivo
for future
therapeutic applications.
14
Another emerging technology is that based on targeting microRNAs (miRNAs),
which are single-stranded nonprotein coding RNAs (21-23 bases in length) that
regulate gene expression in plants and animals.
15,16
Unlike siRNA - mediated RNAi
that inhibits translation of a single gene, an miRNA may target hundreds of differ-
ent mRNAs. The evidence of miRNA participation in a wide range of cellular func-
tions in mammals (e.g. cell growth and apoptosis, heart and brain development and
insulin secretion), together with their involvement in diseases such as neurodegen-
erative disorders and cancer, makes them attractive new druggable targets.
17
Recently, chemically modifi ed anti-miRNA oligonucleotides have proved valuable
tools to specifi cally silence miRNAs
in vivo
, enabling their functions to be unrav-
elled in a new, promising therapeutic strategy.
18,19
Small synthetic oligonucleotides can also be used to target specifi c sequences
of the double helix of DNA (antigene strategy, Figure 9.1).
20
These molecules, called
triplex - forming oligonucleotides (TFOs),
21
recognize and bind polypurine/polypyri-
midine tracts in DNA duplexes through non Watson-Crick hydrogen-bonding
interactions (Hoogsteen bonding) forming a supramolecular structure that contains
three strands. TFOs have been used to selectively inhibit or regulate gene expression
at the transcriptional level, and to correct genetic defects arising from point muta-
tions.
22
Nevertheless, problems such as the low stability of triple helices, the need to
improve their delivery and stability in the intracellular environment and their access
to the highly packed nuclear DNA still have to be suitably addressed.
In addition to interacting with each other through specifi c hydrogen bonding,
structured nucleic acids (DNA and RNA) also bind to a wide range of target
′
