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A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity

By Jesper B. Bramsen, Maria B. Laursen, Anne F. Nielsen, Thomas B. Hansen, Claus Bus, Niels Langkjaer, B. Ravindra Babu, Torben Hojland, Mikhail Abramov, Arthur Van Aerschot, Dalibor Odadžić, Romualdas Smicius, Jens Haas, Cordula Andree, Jharna Barman, Malgorzata Wenska, Puneet Srivastava, Chuanzheng Zhou, Dmytro Honcharenko, Simone Hess, Elke Müller, Georgii V. Bobkov, Sergey N. Mikhailov, Eugenio Fava, Thomas F. Meyer, Jyoti Chattopadhyaya, Marino Zerial, Joachim W. (Prof. Dr.) Engels, Piet Herdewijn, Jesper Wengel and Jørgen Kjems

Abstract

The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity

Topics: ddc:570
Year: 2009
OAI identifier: oai:publikationen.ub.uni-frankfurt.de:7469

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  1. (1997). 1, 5-anhydrohexitol oligonucleotides: synthesis, base pairing and recognition by regular oligodeoxyribonucleotides and oligoribonucleotides.
  2. (2005). A human, ATPindependent, RISC assembly machine fueled by pre-miRNA.
  3. (2007). A novel entry to 20-O-aminopropyl modified nucleosides amenable for further modifications.
  4. (2006). A structural basis for discriminating between self and nonself double-stranded RNAs in mammalian cells.
  5. (2003). Activation of the interferon system by shortinterfering RNAs.
  6. (2005). Activity of stabilized short interfering RNA in a mouse model of hepatitis B virus replication.
  7. (2005). Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation.
  8. (2004). Argonaute2 is the catalytic engine of mammalian RNAi.
  9. (2001). Argonaute2, a link between genetic and biochemical analyses of RNAi.
  10. (2003). Asymmetry in the assembly of the RNAi enzyme complex.
  11. (2006). Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras.
  12. (2006). Chemical modification of siRNAs to improve serum stability without loss of efficacy.
  13. (2006). Cleavage of the siRNA passenger strand during RISC assembly in human cells.
  14. (2007). Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC.
  15. (2006). Conformationally constrained 20-N,40-C-ethylene-bridged thymidine (aza-ENA-T): synthesis, structure, physical, and biochemical studies of aza-ENAT-modified oligonucleotides.
  16. (2004). Conjugate for efficient delivery of short interfering RNA (siRNA) into mammalian cells.
  17. (1999). D-Altritol Nucleic Acids (ANA): Hybridisation properties, stability, and initial structural analysis.
  18. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.
  19. (2007). Evaluation of locked nucleic acid-modified small interfering RNA in vitro and in vivo.
  20. (2006). Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways.
  21. (2007). Five- and six-membered conformationally locked 20,40-carbocyclic ribo-thymidines: synthesis, structure, and biochemical studies.
  22. (2005). Fully 20-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA.
  23. (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.
  24. (2003). Functional siRNAs and miRNAs exhibit strand bias.
  25. (2003). Functionalized LNA (locked nucleic acid): high-affinity hybridization of oligonucleotides containing N-acylated and N-alkylated 20-amino-LNA monomers.
  26. (2004). Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference.
  27. (2007). Improved silencing properties using small internally segmented interfering RNAs.
  28. (2004). In vivo activity of nuclease-resistant siRNAs.
  29. (2006). Innate sensing of self and non-self RNAs by Toll-like receptors.
  30. (2005). Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality.
  31. (2004). Modulation of CpG oligodeoxynucleotide-mediated immune stimulation by locked nucleic acid (LNA).
  32. (1995). Novel linear and branched oligodeoxynucleotide analogues containing 40-C-(hydroxymethyl)thymidine.
  33. (2005). Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes.
  34. (2008). Phosphoramidite building blocks for efficient incorporation of 20-O-aminoethoxy(and propoxy)methyl nucleosides into oligonucleotides.
  35. (2006). Position-specific chemical modification of siRNAs reduces ‘‘off-target’’ transcript silencing.
  36. (2005). Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs.
  37. (2003). Prediction of mammalian microRNA targets.
  38. (2008). Preparation of zwitterionic ribonucleoside phosphoramidites for solid-phase siRNA synthesis.
  39. (2008). R: a language and environment for statistical computing. R Foundation for Statistical Computing,
  40. (2003). RNA interference in mammalian cells by chemically-modified RNA.
  41. (2002). RNAi in human cells: basic structural and functional features of small interfering RNA.
  42. (2005). Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7.
  43. (2002). Single-stranded antisense siRNAs guide target RNA cleavage in RNAi.
  44. (2003). siRNA function in RNAi: a chemical modification analysis.
  45. (2004). Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing in liver cells.
  46. (2005). Structural basis for 50-end-specific recognition of guide RNA by the A. fulgidus Piwi protein.
  47. (2003). Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells.
  48. (2008). Structure of the guide-strand-containing argonaute silencing complex.
  49. (1995). Synthesis and evaluation of oligodeoxynucleotides containing acyclic nucleosides: introduction of three novel analogues and a summary.
  50. (2008). Synthesis of 20-O-modified adenosine building blocks and application for RNA interference.
  51. (2004). Synthesis, physicochemical and biochemical studies of 10,20-oxetane constrained adenosine and guanosine modified oligonucleotides, and their comparison with those of the corresponding cytidine and thymidine analogues.
  52. (2003). The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes.
  53. (2004). Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs.
  54. (2003). Tolerance for mutations and chemical modifications in a siRNA.

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