Preparation of oligoribonucleotides containing 4-thiouridine using Fpmp chemistry. Photo-crosslinking to RNA binding proteins using 350 nm irradiation

Nucleic Acids Res. 1996 Aug 15;24(16):3173-80. doi: 10.1093/nar/24.16.3173.

Abstract

The preparation of a 4-thiouridine phosphoramidite suitable for RNA synthesis and its subsequent incorporation into oligoribonucleotides is described. The thiol group is protected with a 2-cyanoethyl group and the 2'-OH with a 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl function. Thiouridine-containing oligoribonucleotides were used as 350 nm UV crosslinking probes for the photoaffinity labelling of RNA binding proteins. Specific crosslinking was demonstrated between the Rev protein of HIV-1 (as a glutathione S-transferase fusion protein) and its RNA target, the Rev-responsive element. It was not possible to generate crosslinks between the RNA bacteriophage MS2 coat protein and the initiator stem-loop of the replicase gene, to which it binds. These results are consistent with the structural data available on both systems.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Affinity Labels
  • Base Sequence
  • Capsid / chemistry
  • Capsid / metabolism
  • Capsid / radiation effects
  • Capsid Proteins*
  • Gene Products, rev / chemistry
  • Gene Products, rev / genetics
  • Gene Products, rev / metabolism
  • Gene Products, rev / radiation effects
  • HIV-1 / chemistry
  • HIV-1 / genetics
  • HIV-1 / metabolism
  • HIV-1 / radiation effects
  • Molecular Probes
  • Molecular Sequence Data
  • Oligoribonucleotides / chemistry*
  • Oligoribonucleotides / metabolism
  • Oligoribonucleotides / radiation effects
  • Piperidines / chemistry*
  • Protein Binding / radiation effects
  • RNA-Binding Proteins / chemistry*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • RNA-Binding Proteins / radiation effects
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Fusion Proteins / radiation effects
  • Thionucleotides / chemistry*
  • Thionucleotides / metabolism
  • Thionucleotides / radiation effects
  • Thiouridine / chemistry*
  • Ultraviolet Rays
  • rev Gene Products, Human Immunodeficiency Virus

Substances

  • Affinity Labels
  • Capsid Proteins
  • Gene Products, rev
  • Molecular Probes
  • Oligoribonucleotides
  • Piperidines
  • RNA-Binding Proteins
  • Recombinant Fusion Proteins
  • Thionucleotides
  • rev Gene Products, Human Immunodeficiency Virus
  • Thiouridine