Real-time PCR detection of protein analytes with conformation-switching aptamers.
We have developed a novel method that uses conformation-switching aptamers for real-time PCR analysis of protein analytes. The aptamers have been designed so that they assume one secondary structure in the absence of a protein analyte and a different secondary structure in the presence of a protein such as thrombin or platelet-derived growth factor (PDGF). The protein-bound structure in turn assembles a ligation junction for the addition of a real-time PCR primer. Protein concentrations could be specifically detected into the picomolar range, even in the presence of cell lysates. The method has advantages relative to both immunoPCR (because no signal is produced by background binding) and the proximity ligation assay (PLA) (because only one epitope, rather than two epitopes, on a protein surface must be bound).
Yang
Litao
L
Department of Chemistry and Biochemistry, Institute for Cell and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
Ellington
Andrew D
AD
D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001483:Base Sequence; D015949:Factor IXa / Q000032:analysis; D015951:Factor Xa / Q000032:analysis; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D010982:Platelet-Derived Growth Factor / Q000032:analysis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012710:Serum Albumin, Bovine; D013917:Thrombin / Q000032:analysis*
D052157:Aptamers, Nucleotide; D016376:Oligonucleotides, Antisense; D010982:Platelet-Derived Growth Factor; D012710:Serum Albumin, Bovine; D015949:Factor IXa; D013917:Thrombin; D015951:Factor Xa