Fast, reversible interaction of prion protein with RNA aptamers containing specific sequence patterns.
One of the unsolved problems in prion diseases relates to the physiological function of cellular prion protein (PrP), of which a misfolded isoform is the major component of the transmissible spongiform encephalopathies agent. Knowledge of the PrP-binding molecules may help in elucidating its role and understanding the pathological events underlying prion diseases. Because nucleic acids are known to bind PrP, we attempted to identify the preferred RNA sequences that bind to the ovine recombinant PrP. An in vitro selection approach (SELEX) was applied to a pool of 80-nucleotide(nt)-long RNAs containing a randomised 40-nt central region. The most frequently isolated aptamer, RM312, was also the best ligand (20 nM KD value), according to both surface plasmon resonance and filter binding assays. The fast rates of association and dissociation of RM312 with immobilized PrP, which are reminiscent of biologically relevant interactions, could point to a physiological function of PrP towards cellular nucleic acids. The minimal sequence that we found necessary for binding of RM312 to PrP presents a striking similarity with one previously described PrP aptamer of comparable affinity. In addition, we here identify the two lysine clusters contained in the N-terminal part of PrP as its main nucleic-acid binding sites.
Mercey
R
R
Infectiologie Animale et Santé Publique, Institut National de la Recherche Agronomique, Centre de Tours, Nouzilly, France.
Lantier
I
I
Maurel
M-C
MC
Grosclaude
J
J
Lantier
F
F
Marc
D
D
D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D018621:PrPC Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D052156:SELEX Aptamer Technique; D012756:Sheep; D013329:Structure-Activity Relationship
D052157:Aptamers, Nucleotide; D010455:Peptides; D018621:PrPC Proteins