Chemical Strategies to Enhance Antisense Strand Selection and Minimize Off-Target Effect-Mediated by siRNAs

Methods Mol Biol. 2025:2965:479-488. doi: 10.1007/978-1-0716-4742-4_25.

Abstract

Small interfering RNAs (siRNAs) hold significant promise as therapeutic agents for treating human diseases. However, their use is often limited by unintended off-target effects arising from both the sense and antisense strands. Since the 5'-phosphate group on either strand is necessary for recognition by Argonaute 2 (AGO2), the core protein in the RNA-induced silencing complex (RISC), blocking phosphorylation of the sense strand may prevent its incorporation into RISC, thereby reducing potential off-target effects. In this chapter, we describe the use of biotin, a naturally occurring compound, to block 5'-end phosphorylation on either the sense or antisense strand to prevent their selection by AGO2. This strategy should offer a practical option to enhance siRNA specificity and efficacy.

Keywords: Antisense strand; Biotin; Design tools; Off-target effects; Thermodynamics; siRNA.

MeSH terms

  • Argonaute Proteins / metabolism
  • Biotin / chemistry
  • Humans
  • Phosphorylation / drug effects
  • RNA Interference
  • RNA, Small Interfering* / chemistry
  • RNA, Small Interfering* / genetics
  • RNA, Small Interfering* / metabolism
  • RNA-Induced Silencing Complex / metabolism

Substances

  • RNA, Small Interfering
  • Argonaute Proteins
  • Biotin
  • AGO2 protein, human
  • RNA-Induced Silencing Complex