TY - JOUR
T1 - Promiscuous 8-alkoxyadenosines in the guide strand of an SiRNA
T2 - Modulation of silencing efficacy and off-pathway protein binding
AU - Ghanty, Uday
AU - Fostvedt, Erik
AU - Valenzuela, Rachel
AU - Beal, Peter A.
AU - Burrows, Cynthia J.
PY - 2012/10/24
Y1 - 2012/10/24
N2 - 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.
AB - 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.
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U2 - 10.1021/ja307102g
DO - 10.1021/ja307102g
M3 - Article
C2 - 23030736
AN - SCOPUS:84867795819
VL - 134
SP - 17643
EP - 17652
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
SN - 0002-7863
IS - 42
ER -