Structure-Guided Control of siRNA Off-Target Effects

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Development

Background:

• Short interfering RNAs (siRNAs) are valuable therapeutics utilizing the RNA interference (RNAi) pathway.
• Chemical modifications are essential to overcome structural limitations of native siRNAs for drug development.
• Structural insights into the human RNAi pathway facilitate the optimization of siRNA properties.

Purpose of the Study:

• To determine the structure of human Argonaute 2 (hAgo2) bound to a modified siRNA.
• To investigate how a triazolyl nucleotide at position 1 (g1) affects siRNA binding and function.
• To explore structure-guided modifications for enhancing siRNA selectivity and potency.

Main Methods:

• X-ray crystallography to determine the 2.3 Å resolution structure of hAgo2 complexed with a g1-modified siRNA.
• Biochemical assays to measure the binding affinity of modified siRNAs to target RNAs.
• Functional assays to assess on-target and off-target knockdown efficacy.

Main Results:

• The triazolyl nucleotide analogue inserts into the hAgo2 central RNA binding cleft, modulating guide-target RNA pairing.
• Modification significantly reduced binding affinity for seed-only matched targets but not fully matched targets.
• siRNA potency for off-target repression was reduced (4-fold increase in IC50), while on-target knockdown was improved (2-fold reduction in IC50).

Conclusions:

• Introducing unnatural nucleotides like triazolyl analogues into siRNAs offers a structure-based approach to control target binding and potency.
• This modification enhances siRNA selectivity by reducing off-target effects while improving on-target gene silencing.
• Projection of substituent groups into the hAgo2 central cleft provides a novel strategy for developing more selective siRNA therapeutics.