A three-dimensional design strategy for a protein-responsive shRNA switch

Methods Mol Biol. 2014;1111:269-86. doi: 10.1007/978-1-62703-755-6_20.

Abstract

We have recently developed synthetic short hairpin RNA (shRNA) switches that respond to intracellular proteins and control the expression of target genes in mammalian cells (Kashida et al. Nucleic Acids Res 40:9369-9378, 2012; Saito et al. Nat Commun 2:160, 2011). Here, we describe a method for the three-dimensional (3D) design of a protein-responsive shRNA switch that employs modeling software and known 3D structures of RNA-protein complexes. We were able to predict the effect of steric hindrance between the Dicer enzyme and shRNA-binding protein in silico by superimposing the 3D model of the shRNA switch on Dicer. The function of the designed switch can be evaluated in vitro and in living cells. Our expertise will help utilize the 3D structure of biomacromolecular complexes for the design of functional genetic switches.

MeSH terms

  • Animals
  • Base Sequence
  • Cell Line
  • Computer Simulation
  • DEAD-box RNA Helicases / genetics
  • DEAD-box RNA Helicases / metabolism*
  • Green Fluorescent Proteins / genetics
  • Humans
  • Models, Molecular
  • Protein Binding
  • RNA Interference
  • RNA, Small Interfering / chemistry
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Ribonuclease III / genetics
  • Ribonuclease III / metabolism*
  • Software
  • Transcription, Genetic

Substances

  • RNA, Small Interfering
  • Recombinant Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • DICER1 protein, human
  • Ribonuclease III
  • DEAD-box RNA Helicases