Synthetic Dicer-Substrate siRNAs as Triggers of RNA Interference - RNA Interference from Biology to Therapeutics - page 39

( AKT1 ), the DsiRNA and 21-nt siRNA showed a similar duration of action. It is not
thought that the DsiRNA design conveys any specific benefit for duration of action;
rather, it appears that duration of action is largely dependent on the potency of the
silencing reagent and that for 4/5 of the cases studied here, the DsiRNAs were more
potent and therefore showed extended silencing over time. The time course of
silencing for the anti- TP53 DsiRNA and 21-nt siRNA are shown in Fig. 2.4b .

2.2.2 Chemical Modi fi cation of DsiRNAs

2.2.2.1 Chemical Modi fi cation and Nuclease Stability

Synthetic nucleic acids are readily degraded by nucleases present in serum and in
cells. In serum, the primary activity of concern is a 3¢ -exonuclease [ 38 ] , whereas in
cell extracts endonucleases appear to play a greater role. Fortunately, antisense oli-
gonucleotides (ASOs), siRNAs, and DsiRNAs can be made using chemical
modifications which impart nuclease stability as well as improve their safety profiles
and general pharmacodynamic properties. Several comprehensive reviews of this
topic have recently been published, and readers are referred to these sources for
more details [ 39- 41 ] .
It is possible to heavily modify an RNA duplex so that it is almost completely
resistant to nuclease attack. Unfortunately, many of the modifications that convey
nuclease resistance also reduce the potency of the siRNA, presumably by altering
interactions with the various proteins that mediate RNAi in cells, such as Dicer and
Argonaute 2 (Ago2). In general, chemical modifications that impart nuclease resis-
tance either involve the internucleoside phosphate bonds or the sugar backbone of
the nucleic acid. The phosphorothioate modification (PS) substitutes sulfur for a
non-bridging oxygen in the phosphate internucleoside linkage. This conveys relative
resistance to many nucleases but can also make the oligonucleotide more “sticky” to
proteins and can significantly alter function in undesired ways [ 42 ] . While it is com-
mon practice to completely modify ASOs with PS bonds, this modification is usu-
ally used sparingly in siRNAs. SiRNAs with high PS content can perform poorly,
and altered interactions between the RNA and the protein machinery in RISC may
be implicated [ 43 ]. Selective incorporation of PS linkages near the ends of the duplex
and, particularly in the 3¢-overhangs, helps protect these vulnerable sites and is a
modification strategy commonly used today [ 40 ] . Single-stranded RNA domains,
such as the siRNA 3¢-overhangs, are highly susceptible to degradation. An inverted
dT base, PS bonds, or other modifications are often placed at this site.
Modi fi cation at the 2 ¢-position of the ribose usually decreases the susceptibility
of the neighboring phosphate bonds to nuclease attack, and a wide variety of
2¢-modified residues are routinely employed to modify siRNAs. In particular,
2¢ - O -methyl (2 ¢-OMe) RNA is a naturally occurring chemical modification that is
found in mammalian tRNAs and rRNAs. This modification is relatively inexpen-
sive to incorporate into synthetic nucleic acids and has no known toxicity. Other