NR AOWT
AU Mercey,R.; Marc,D.; Vaudin,P.; Maurel,M.C.; Lantier,I.; Lantier,F.
TI Isolation and characterization of RNA ligands to recombinant ovine prion protein
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - DG-68
PT Konferenz-Poster
AB
Search for molecules binding to the prion protein may help understand the enigmatic nature of the TSE agent, as well as the physiological role of the protein.
We attempted to identify RNAs that bind recombinant ovine PrP in vitro. An in vitro selection approach (SELEX) was applied to a pool of 80 nucleotide-long RNAs containing a 40-nt central region of random sequence. Four independent selections of 10 to 15 cycles were carried out, and more than 150 clones were sequenced. These were grouped in four families and nine pairs sharing identical or similar motifs. The largest family contained sequences identified in three independent series of selection. None of our aptamers contained the guanine-quadruplex motif identified by Weiss et al. (1997), although we verified that an RNA containing such a motif strongly bound to recombinant PrP in vitro.
The best ligands showed a 10 nM dissociation constant, as determined through surface plasmon resonance (Biacore system). Systematic searches in the public databases revealed that the nucleotide motifs identified in the best ligands were unfrequent in the mammals sequences. However, the interaction between PrP and aptamers is likely structure-dependent rather than sequence-dependent, and analysis of the sequences did not allow us to unambiguously identify RNA structure motifs involved in that interaction.
Further work is in progress towards identifying the structural motifs in the RNA aptamers, as well as identifying natural RNA ligands of the prion protein. Whatever their biological relevance, these RNA aptamers may enlarge the repertoire of PrP-specific ligands with potential usefulness in the detection of PrP.
AD Raphael Mercey, Daniel Marc, Pascal Vaudin, Marie-Christine Maurel, Isabelle Lantier, Frédéric Lantier, INRA - Centre de Tours, France
SP englisch
PO Deutschland