2 research outputs found
Promiscuous 8‑Alkoxyadenosines in the Guide Strand of an SiRNA: Modulation of Silencing Efficacy and Off-Pathway Protein Binding
8-Alkoxyadenosines have the potential to exist in anti
or syn conformations
around the glycosidic bond when paired opposite to U or G in the complementary
strands, thereby placing the sterically demanding 8-alkoxy groups
in the major or minor groove, respectively, of duplex RNA. These modified
bases were used as “base switches” in the guide strands
of an siRNA to prevent off-pathway protein binding during delivery
via placement of the alkoxy group in the minor groove, while maintaining
significant RNAi efficacy by orienting the alkoxy group in the major
groove. 8-Alkoxyadenosine phosphoramidites were synthesized and incorporated
into the guide strand of caspase 2 siRNA at four different positions:
two in the seed region, one at the cleavage junction, and another
nearer to the 3′-end of the guide strand. Thermal stabilities
of the corresponding siRNA duplexes showed that U is preferred over
G as the base-pairing partner in the complementary strand. When compared
to the unmodified positive control siRNAs, singly modified siRNAs
knocked down the target mRNA efficiently and with little or no loss
of efficacy. Doubly modified siRNAs were found to be less effective
and lose their efficacy at low nanomolar concentrations. SiRNAs singly
modified at positions 6 and 10 of the guide strand were found to be
effective in blocking binding to the RNA-dependent protein kinase
PKR, a cytoplasmic dsRNA-binding protein implicated in sequence-independent
off-target effects
Structure-Guided Control of siRNA Off-Target Effects
Short interfering
RNAs (siRNAs) are promising therapeutics that
make use of the RNA interference (RNAi) pathway, but liabilities arising
from the native RNA structure necessitate chemical modification for
drug development. Advances in the structural characterization of components
of the human RNAi pathway have enabled structure-guided optimization
of siRNA properties. Here we report the 2.3 Ă… resolution crystal
structure of human Argonaute 2 (hAgo2), a key nuclease in the RNAi
pathway, bound to an siRNA guide strand bearing an unnatural triazolyl
nucleotide at position 1 (g1). Unlike natural nucleotides, this analogue
inserts deeply into hAgo2’s central RNA binding cleft and thus
is able to modulate pairing between guide and target RNAs. The affinity
of the hAgo2–siRNA complex for a seed-only matched target was
significantly reduced by the triazolyl modification, while the affinity
for a fully matched target was unchanged. In addition, siRNA potency
for off-target repression was reduced (4-fold increase in IC<sub>50</sub>) by the modification, while on-target knockdown was improved (2-fold
reduction in IC<sub>50</sub>). Controlling siRNA on-target versus
microRNA (miRNA)-like off-target potency by projection of substituent
groups into the hAgo2 central cleft from g1 is a new approach to enhance
siRNA selectivity with a strong structural rationale