Article thumbnail

A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects

By Jesper B. Bramsen, Malgorzata M. Pakula, Thomas B. Hansen, Claus Bus, Niels Langkjær, Dalibor Odadzic, Romualdas Smicius, Suzy L. Wengel, Jyoti Chattopadhyaya, Joachim W. Engels, Piet Herdewijn, Jesper Wengel and Jørgen Kjems


Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2–8 of either siRNA strand counting from the 5′-end) and complementary sequences in the 3′UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found several differently modified siRNAs to exercise reduced off-targeting yet incorporation of the strongly destabilizing unlocked nucleic acid (UNA) modification into position 7 of the siRNA most potently reduced off-targeting for all tested sequences. Notably, such position-specific destabilization of siRNA–target interactions did not significantly reduce siRNA potency and is therefore well suited for future siRNA designs especially for applications in vivo where siRNA concentrations, expectedly, will be low

Topics: Molecular Biology
Publisher: Oxford University Press
OAI identifier:
Provided by: PubMed Central

To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.

Suggested articles


  1. (1997). 10,5 0-anhydrohexitol oligonucleotides: synthesis, base pairing and recognition by 5772
  2. (2006). 30 UTR seed matches, but not overall identity, are associated with RNAi off-targets.
  3. (2009). A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity.
  4. (2005). A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites.
  5. (1999). Altritol Nucleic Acids (ANA): hybridisation properties, stability, and initial structural analysis.
  6. (2009). Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 50-terminal phosphorylation both in vitro and in vivo.
  7. (2010). Application of small interfering RNAs modified by unlocked nucleic acid (UNA) to inhibit the heart-pathogenic coxsackievirus B3.
  8. (2005). Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation.
  9. (2007). Comparison of siRNA-induced off-target RNA and protein effects.
  10. (2006). Conformationally constrained 20-N,40-C-ethylene-bridged thymidine (aza-ENA-T): synthesis, structure, physical, and biochemical studies of aza-ENA-T-modified oligonucleotides.
  11. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.
  12. (2003). Expression profiling reveals off-target gene regulation by RNAi.
  13. (2008). Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect.
  14. (2007). Improved silencing properties using small internally segmented interfering RNAs.
  15. (2006). Induction of the interferon response by siRNA is cell type- and duplex length-dependent.
  16. (2008). Knockdown of TNFR1 by the sense strand of an ICAM-1 siRNA: dissection of an off-target effect.
  17. (2004). LNA (locked nucleic acid): high-affinity targeting of complementary
  18. (2005). Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality.
  19. (2006). mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes.
  20. (2004). Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs).
  21. (1995). Novel linear and branched oligodeoxynucleotide analogues containing 40-C-(hydroxymethyl)thymidine.
  22. (2006). Off-target effects by siRNA can induce toxic phenotype.
  23. (2008). Off-target effects of siRNA specific for GFP.
  24. (2005). Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes.
  25. (2006). Position-specific chemical modification of siRNAs reduces ‘‘off-target’’ transcript silencing.
  26. (2008). Preparation of zwitterionic ribonucleoside phosphoramidites for solid-phase siRNA synthesis.
  27. (2005). Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation.
  28. (2006). RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle system.
  29. (2006). RUNNING INTERFERENCE: prospects and obstacles to using small interfering RNAs as small molecule drugs.
  30. (2005). Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7.
  31. (2005). siRNA-mediated off-target gene silencing triggered by a 7nt complementation.
  32. (2003). siRNAs can function as miRNAs.
  33. (2003). Specificity of short interfering RNA determined through gene expression signatures.
  34. (2008). Structure of the guide-strand-containing argonaute silencing complex.
  35. (2008). Synthesis of 20-O-modified adenosine building blocks and application for RNA interference.
  36. (2008). The impact of microRNAs on protein output.
  37. (2009). UNA (unlocked nucleic acid): a flexible RNA mimic that allows engineering of nucleic acid duplex stability.
  38. (2010). Utilization of unlocked nucleic acid (UNA) to enhance siRNA performance in vitro and in vivo.
  39. (2008). Widespread changes in protein synthesis induced by microRNAs.
  40. (2006). Widespread siRNA ‘‘off-target’’ transcript silencing mediated by seed region sequence complementarity.