Thioaptamer interactions with prion proteins: sequence-specific and non-specific binding sites.
Binding of nucleic acids to the prion protein (PrP) created a conundrum that required distinguishing between non-specific interactions and biologically important polynucleotides. In the process of developing selective ligands for PrP, we found using a single-stranded DNA thioaptamer library that the binding of thioaptamers to PrP occurs on at least two different sites on the protein. Selection against recombinant (rec) PrP of Syrian hamster (SHa) sequence 90-231 folded into an alpha-helical-rich conformation identified a 12-base consensus sequence within a series of 20 thioaptamers, all of which consist of 40 bases. Each thioaptamer was comprised of both normal and thio-dA modified bases. One thioaptamer designated 97 bound to recSHaPrP with affinity of 0.58(+/-0.1) nM; lower affinities for bovine (Bo), and human (Hu) were found, establishing that binding is dependent on the primary structure of PrP. High affinity binding of thioaptamer 97 to PrP was found to be mediated through the dodecyl sequence GACACAAGCCGA within the consensus region with five critical backbone modifications 5' to each dA residue. A control oligonucleotide with an equivalent number of phosphorothioates to thioaptamer 97 and a scrambled consensus sequence could not distinguish among the three PrP sequences. Control oligonucleotides bearing non-selected sequences bound to PrP at a sequence-independent DNA-binding site. In contrast, the high-affinity binding of thioaptamer 97 to PrP depends on (1) backbone modifications, (2) oligonucleotide sequence, and (3) PrP sequence.
King
David J
DJ
Institute for Neurodegenerative Diseases, University of California San Francisco, CA 94143-0518, USA.
Safar
Jiri G
JG
Legname
Giuseppe
G
Prusiner
Stanley B
SB
D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D020650:Combinatorial Chemistry Techniques; D006224:Cricetinae; D006801:Humans; D008969:Molecular Sequence Data; D011328:Prions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012756:Sheep; D013873:Thionucleotides / Q000737:chemistry* / Q000378:metabolism
D052157:Aptamers, Nucleotide; D011328:Prions; D013873:Thionucleotides