title: Fluorescence anisotropy reduction of allosteric aptamer for sensitive and specific protein signaling.
abstract: Real time protein signaling in a complex medium may provide a promising way for high-throughput protein analysis, but it is largely unmet due to the challenge of signal transduction and the interferences of nonspecific binding and high background. Our recent work indicates that a fluorescent aptamer can display a protein binding-induced reduction of fluorescence anisotropy (FA) (Zhang, D.; Lu, M.; Wang, H. J. Am. Chem. Soc. 2011, 133, 9188-9191), which is exclusively different from a traditionally simplified concept hinting a molecular size increase-induced FA increase. Inspired by this unexpected observation, we describe a novel FA reduction approach for protein signaling. The feasibility of this approach is demonstrated through the assays of a blood protein human α-thrombin and an oncoprotein human platelet-derived growth factor B-chain (PDGF-BB) using two screened fluorescent aptamers, respectively. By the developed FA reduction method, the spiked human α-thrombin in diluted serum can be detected at the concentration as low as 250 pM. In contrast, in a traditional molecular size-dependent FA assay, the thrombin spiked in diluted serum cannot induce reliable FA change even at a 256-fold higher concentration (64 nM). The results clearly show that the FA reduction approach has a dramatically enhanced specificity against target protein and high sensitivity in complex medium and is applicable to the no-separation based detection of proteins in biological matrixes.
authors.forename: Dapeng
authors.initials: D
authors.lastname: Zhang
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhao
authors.forename: Bailin
authors.initials: B
authors.lastname: Zhao
authors.forename: Hailin
authors.initials: H
authors.lastname: Wang
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001681:Biological Assay / Q000379:methods*; D002417:Cattle; D005454:Fluorescence Polarization / Q000379:methods*; D006801:Humans; D011485:Protein Binding; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D052157:Aptamers, Nucleotide; D013917:Thrombin