Real-time rolling circle amplification for protein detection.
Real-time nucleic acid amplification methods can be extremely useful for the identification and quantitation of nucleic acid analytes, but are more difficult to adapt to protein or other analytes. To facilitate the development of real-time rolling circle amplification (RCA) for protein targets, we have developed a novel type of conformation-switching aptamer that can be circularized upon interaction with its protein target, the platelet-derived growth factor (PDGF). Using the structure-switching aptamer, real-time RCA can be used to specifically quantitate PDGF down to the low-nanomolar range (limit of detection, 0.4 nM), even against a background of cellular lysate. The aptamer can also be adapted to RCA on surfaces, although quantitation proved to be more difficult. One of the great advantages of the method described herein is that it can be immediately adapted to almost any aptamer and does not require two or more affinity reagents as do sandwich or proximity assays.
Yang
Litao
L
Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA.
Fung
Christine W
CW
Cho
Eun Jeong
EJ
Ellington
Andrew D
AD
D052157:Aptamers, Nucleotide / Q000737:chemistry; D001483:Base Sequence; D005347:Fibroblasts / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010982:Platelet-Derived Growth Factor / Q000032:analysis*; D013499:Surface Properties; D013997:Time Factors
D052157:Aptamers, Nucleotide; D010982:Platelet-Derived Growth Factor