Related Experiment Video

Updated: Aug 20, 2026

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro
09:16

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

Published on: May 3, 2014

Antisense-induced Fas mRNA degradation produces site-specific stable 3'-mRNA fragment by exonuclease cleavage at the

Håkan Thonberg1, Cecilia Dahlgren, Claes Wahlestedt

  • 1Center for Genomics and Bioinformatics, Karolinska Institutet, 171 77 Stockholm, Sweden. hakan.thonberg@cgb.ki.se

Oligonucleotides
|January 1, 2005
PubMed

Insights

Researchers compared phosphorothioate oligonucleotides for their ability to degrade target messenger RNA (mRNA) using RNase H. Four antisense oligonucleotides significantly reduced Fas mRNA levels in mouse cells, with one causing a specific cleavage fragment.

Area of Science:

  • Molecular Biology
  • Antisense Technology
  • RNA Degradation

Background:

  • Antisense-mediated mRNA degradation relies on the nuclease RNase H.
  • RNase H specifically cleaves RNA in RNA-DNA duplexes.
  • Phosphorothioate oligonucleotides are utilized as antisense agents.

Purpose of the Study:

  • To compare the efficacy of six different phosphorothioate oligonucleotides.
  • To assess their ability to induce target-specific mRNA degradation.
  • To investigate the mechanism of mRNA cleavage by RNase H.

Main Methods:

  • Cultured mouse AML12 cells were treated with antisense oligonucleotides.
  • Target mRNA (Fas receptor) levels were analyzed using Northern blotting.
  • Ribonuclease protection assay (RPA) was employed to characterize cleavage products.

Main Results:

  • Four out of six tested antisense oligonucleotides significantly reduced Fas mRNA levels.
  • A specific 3' cleavage fragment of Fas mRNA was identified by RPA.
  • The cleavage site was mapped to the region complementary to the antisense oligonucleotide.

Conclusions:

  • Phosphorothioate oligonucleotides can effectively induce target-specific mRNA degradation via RNase H.
  • The study identified effective antisense sequences for Fas mRNA.
  • RPA is a valuable tool for characterizing mRNA cleavage products and mapping cleavage sites.

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