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

Abstract

Aptamer-based fluorescence anisotropy (FA) assay combines the advantages of affinity aptamers in good stability, easy generation, and facile labeling and the benefits of FA in homogeneous analysis, such as robustness, simplicity, and high reproducibility. By using a fluorophore-labeled aptamer, FA detection of a small molecule is not as easy as detection of protein because the binding of a small molecule cannot cause significant increase of molecular weight of the dye-labeled aptamer. The intramolecular interaction between labeled tetramethylrhodamine (TMR) and DNA aptamer bases dramatically affects the local rotation and FA of TMR. This intramolecular interaction can be altered by aptamer conformation change upon target binding, leading to a significant change of FA of TMR. Taking this unique feature of a TMR-labeled aptamer, we described a noncompetitive aptamer-based fluorescence anisotropy assay for detection of small molecules by using ochratoxin A (OTA) as a model. We successfully identified the specific TMR-labeling sites of aptamers with sensitive FA response to OTA from the 5′-end, 3′-end and the internal thymine (T) bases. The aptamer with a TMR labeled on the 10th T base exhibited a remarkable FA reduction response to OTA (Δr = 0.078), without requiring any proteins or nanomaterials as FA signal enhancers. This FA approach for OTA showed high sensitivity with a detection limit of 3 nM, a dynamic range from 3 nM to 3 μM, and good selectivity over the tested compounds with similar structures to OTA. The new strategy allowed the detection of OTA in diluted red wine and urine samples. F luorescence anisotropy (FA) is a powerful and unique analytical technique for molecular interactions study and assay developments, which relies on the rotational diffusion of fluorescent tracers or probes under polarized excitation light. 1−5Fluorescence anisotropy shows strength in real-time analysis, simplicity, robustness, and sensitivity. FA assays have been applied to the studies about protein−protein interaction and protein−DNA interaction and the immunoassays for drug discovery, diagnostics, environmental monitoring, and food analysis. 1−7The emergence of aptamers as affinity ligands makes the aptamer-based FA assay attractive due to their advantages over antibodies in ease of synthesis and labeling, great stability, good selectivity, and high binding affinity. 8−12 Target-dependent allosteric structural change is one unique feature of an aptamer, and it allows the construction of an aptamer switch for target analysis through the adaptive transition converting a binding event into a detectable signal. 8,13−17 The applications of fluorophore-labeled aptamers to direct FA analysis of proteins are relatively straightforward as aptamers are usually smaller than proteins in size. 5,8,11,12 The binding of the fluorophorelabeled aptamers to proteins can cause enhanced FA signals by restricting the rotational diffusion of the labeled fluorophore as a result of increased molecular size. 18−23 In contrast, the ...

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

“…gold nanoparticles, graphene, and silica nanoparticles) have been introduced in assay development by increasing the binding-induced molecular weight change (Cruz-Aguado and Penner, 2008;Cui et al, 2012;Huang et al, 2012;Liu et al, 2013;Ye, and Yin 2008;Yu et al, 2013;Zhu et al, 2011Zhu et al, , 2012 Recently, we have reported a simple and noncompetitive FA strategy for a small molecule, ochratoxin A, by using a TMR-labeled aptamer. (Zhao et al, 2014) It is based on the target-binding induced change of intramolecular interactions between TMR and the guanine (G) bases of the aptamer. The interaction significantly affects local rotation of TMR and FA of TMR.…”

“…In this FA strategy, TMR-labeling position of the aptamers plays a crucial role. The intramolecular interaction between TMR and G bases is distance dependent, and TMR labeling may influence the binding affinity of aptamer depending on the labeling sites (Zhao et al, 2014). An ideal labeling position allows TMR-labeled aptamer showing FA change upon target without loss in binding affinity.…”

“…An ideal labeling position allows TMR-labeled aptamer showing FA change upon target without loss in binding affinity. Previously, only terminal ends and T base of aptamers could be examined for TMR labeling in FA analysis of ochratoxin A (Zhao et al, 2014). However, G base in aptamers may not be surrounded by T bases or terminal ends and some aptamers may have less T bases.…”

Different Applications of Magnetic Nanoparticles in the Rapid Monitoring of Ochratoxin A

“…ELISA (enzyme-linked immunosorbent assay) traditionally utilize the antibodies, but its storage is hard, and its preparation needs to long time and high cost 35 . As a new technique with excellent sensitivity for detection of OTA, Yao et al combined the magnetic purification with the system of rolling circular amplification (RCA) to reduce the noise and to enhance the signal.…”

Magnetic Nanoparticles-based Aptasensor Using Gold Nanoparticles as Colorimetric Probes for the Detection of Salmonella typhimurium

“…11,12 Compared with conventional antibodies, aptamers show many advantages, such as more chemically stable, inexpensive, simple to be chemically modified, and so on. 13 This made aptamer probes more flexible in design various types of biosensors, including fluorescence, 14,15 chemiluminescence, 16,17 electrochemical, 18,19 or colorimetric sensors. 20,21 Among these, colorimetry aptasensor, especially gold nanoparticles based colorimetry aptasensor, has attracted much attention owing to their conveniences of visual observation and simple operations.…”