In vitro selection of RNA molecules that inhibit the activity of ricin A-chain

J Biol Chem. 2000 Feb 18;275(7):4937-42. doi: 10.1074/jbc.275.7.4937.

Abstract

The cytotoxin ricin disables translation by depurinating a conserved site in eukaryotic rRNA. In vitro selection has been used to generate RNA ligands (aptamers) specific for the catalytic ricin A-chain (RTA). The anti-RTA aptamers bear no resemblance to the normal RTA substrate, the sarcin-ricin loop (SRL), and were not depurinated by RTA. An initial 80-nucleotide RNA ligand was minimized to a 31-nucleotide aptamer that contained all sequences and structures necessary for interacting with RTA. This minimal RNA formed high affinity complexes with RTA (K(d) = 7.3 nM) which could compete directly with the SRL for binding to RTA. The aptamer inhibited RTA depurination of the SRL and could partially protect translation from RTA inhibition. The IC(50) of the aptamer for RTA in an in vitro translation assay is 100 nM, roughly 3 orders of magnitude lower than a small molecule inhibitor of ricin, pteroic acid, and 2 orders of magnitude lower than the best known RNA inhibitor. The novel anti-RTA aptamers may find application as diagnostic reagents for a potential biological warfare agent and hold promise as scaffolds for the development of strong ricin inhibitors.

MeSH terms

  • Base Sequence
  • Biosensing Techniques
  • DNA Primers
  • Ligands
  • Nucleic Acid Conformation
  • RNA / chemistry
  • RNA / pharmacology*
  • Ricin / antagonists & inhibitors*
  • Ricin / pharmacology

Substances

  • DNA Primers
  • Ligands
  • RNA
  • Ricin