Aptamer-based analysis of angiogenin by fluorescence anisotropy.
Recognition and monitoring proteins in real time and in homogeneous solution has always been a difficult task. Here, we introduce a signal transduction strategy for quick protein recognition and real-time quantitative analysis in homogeneous solutions based on a high-affinity aptamer for protein angiogenin (Ang). The method takes advantage of the sensitive anisotropy signal change of fluorophore-labelled aptamer upon protein/aptamer binding. When the labelled aptamer is bound with its target protein Ang, the increased molecular weight causes the rotational motion of the fluorophore attached to the complex to become much slower. Therefore, increasing the amount of Ang results in a raised anisotropy value of the Ang/aptamer. By monitoring the anisotropy change, we are able to detect the binding events between the aptamer and Ang, and measure Ang concentration quantitatively in homogeneous solutions. This assay is highly selective, with a detection limit of 1 nM of Ang. The dissociation constant of the Ang/aptamer binding is determined in the nanomolar range and changes with increasing salt concentration. One can also use our assay to compare the binding affinities of different ligands for the target molecule. Ang in serum samples of malignant lung cancer was also detected. Efficient protein detection using aptamer-based fluorescence anisotropy measurements is expected to find wide applications in protein monitoring, cancer diagnosis, drug screening and other fields.
Li
Wei
W
State Key Laboratory of Chemo/Biosensing and Chemometrics, Engineering Center for Biomedicine, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China.
Wang
Kemin
K
Tan
Weihong
W
Ma
Changbei
C
Yang
Xiaohai
X
D016880:Anisotropy; D052157:Aptamers, Nucleotide; D001667:Binding, Competitive; D005456:Fluorescent Dyes; D006801:Humans; D011994:Recombinant Proteins / Q000032:analysis; D012259:Ribonuclease, Pancreatic / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000379:methods
D052157:Aptamers, Nucleotide; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; C046366:angiogenin; D012259:Ribonuclease, Pancreatic