Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing (original) (raw)

  1. Aimee L. Jackson1,
  2. Julja Burchard1,
  3. Devin Leake2,
  4. Angela Reynolds2,
  5. Janell Schelter1,
  6. Jie Guo1,
  7. Jason M. Johnson1,
  8. Lee Lim1,
  9. Jon Karpilow2,
  10. Kim Nichols2,
  11. William Marshall2,
  12. Anastasia Khvorova2, and
  13. Peter S. Linsley1
  14. 1Rosetta Inpharmatics, LLC, a wholly owned subsidiary of Merck & Co., Inc., Seattle, Washington 98109, USA
  15. 2Dharmacon Inc., Lafayette, Colorado 80026, USA

Abstract

Transfected siRNAs regulate numerous transcripts sharing limited complementarity to the RNA duplex. This unintended (“off-target”) silencing can hinder the use of RNAi to define gene function. Here we describe position-specific, sequence-independent chemical modifications that reduced silencing of partially complementary transcripts by all siRNAs tested. Silencing of perfectly matched targets was unaffected by these modifications. The chemical modification also reduced off-target phenotypes in growth inhibition studies. Key to the modification was 2′-_O_-methyl ribosyl substitution at position 2 in the guide strand, which reduced silencing of most off-target transcripts with complementarity to the seed region of the siRNA guide strand. The sharp position dependence of 2′-_O_-methyl ribosyl modification contrasts with the broader position dependence of base-pair substitutions within the seed region, suggesting a role for position 2 of the guide strand distinct from its effects on pairing to target transcripts.

Footnotes