TY - JOUR
T1 - Structure-Guided Control of siRNA Off-Target Effects
AU - Suter, Scott R.
AU - Sheu-Gruttadauria, Jessica
AU - Schirle, Nicole T.
AU - Valenzuela, Rachel
AU - Ball-Jones, Alexi A.
AU - Onizuka, Kazumitsu
AU - Macrae, Ian J.
AU - Beal, Peter A.
N1 - Funding Information:
P.A.B. acknowledges support from the National Institutes of Health in the form of Grant R01 GM080784. I.J.M. acknowledges support from National Institutes of Health Grants R01 GM104475 and R21 CA201861. J.S.-G. was supported by
Publisher Copyright:
© 2016 American Chemical Society.
PY - 2016/7/20
Y1 - 2016/7/20
N2 - 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 Å 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 IC50) by the modification, while on-target knockdown was improved (2-fold reduction in IC50). 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.
AB - 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 Å 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 IC50) by the modification, while on-target knockdown was improved (2-fold reduction in IC50). 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.
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U2 - 10.1021/jacs.6b06137
DO - 10.1021/jacs.6b06137
M3 - Article
C2 - 27387838
AN - SCOPUS:84979234392
SN - 0002-7863
VL - 138
SP - 8667
EP - 8669
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 28
ER -