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 bindinginduced 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.

Aptamer fluorescence anisotropy sensors for adenosine triphosphate by comprehensive screening tetramethylrhodamine labeled nucleotides

“…The anti-ATP aptamer can form a hairpin structure when the aptamer binds to ATP, bringing a conformation change (Lin and Patel, 1997). The binding of ATP possibly alters the interaction between TMR and G bases, which affects FA of TMR (Heinlein et al, 2003;Unruh et al, 2005;Yabuki et al, 2003;Zhang et al, 2012), leading to FA changes of TMR. When ATP binding weakens TMR-G interaction, local rotation of labeled TMR increases.…”

Screening interaction between ochratoxin A and aptamers by fluorescence anisotropy approach

“…Fluorescence anisotropy (FA)/fluorescence polarization is a powerful technique, and is not only useful for rapid and quantitative measurements of diverse molecular interactions but also for development of rapid assays [13][14][15][16][17][18][19]. FA analysis is a real-time solutionbased equilibrium method without the need for separation, showing unique advantage of no disturbance in the reaction equilibrium.…”

Identification of Allosteric Nucleotide Sites of Tetramethylrhodamine-Labeled Aptamer for Noncompetitive Aptamer-Based Fluorescence Anisotropy Detection of a Small Molecule, Ochratoxin A

“…35−41 This intramolecular interaction restricts the local rotation of TMR on ssDNA and decreases the fluorescence lifetime of TMR, which affect the FA signal. 20,22,38,42,43 It is also known that the binding of target usually induces an adaptive conformation change of the aptamer. 13−17 This conformational change can bring a subsequent alteration of the interaction between labeled TMR and the bases of the aptamer, and the FA of the labeled TMR on the aptamer can be significantly changed upon target binding.…”

“…The unique intramolecular interaction between TMR and guanine bases and the allosteric property of the aptamer provide another opportunity to develop a simple FA strategy for target analysis with TMR-labeled aptamer, differing from the mass change dependent FA strategy. 5,19,20,22,42,43 Considering that the binding of a small target cannot cause a large change of the molecular mass of the dye-labeled aptamer, this intramolecular interaction dependent FA strategy is more adaptable to small-molecule detection with a dye-labeled aptamer.…”