Single-Stranded DNA Binding Protein-Assisted Fluorescence Polarization Aptamer Assay for Detection of Small Molecules

Abstract

Here, we describe a new fluorescence polarization aptamer assay (FPAA) strategy which is based on the use of the single-stranded DNA binding (SSB) protein from Escherichia coli as a strong FP signal enhancer tool. This approach relied on the unique ability of the SSB protein to bind the nucleic acid aptamer in its free state but not in its target-bound folded one. Such a feature was exploited by using the antiadenosine (Ade)−DNA aptamer (Apt-A) as a model functional nucleic acid. Two fluorophores (fluorescein and Texas Red) were introduced into different sites of Apt-A to design a dozen fluorescent tracers. In the absence of the Ade target, the binding of the labeled aptamers to SSB governed a very high fluorescence anisotropy increase (in the 0.130−0.200 range) as the consequence of (i) the large global diffusion difference between the free and SSB-bound tracers and (ii) the restricted movement of the dye in the SSB-bound state. When the analyte was introduced into the reaction system, the formation of the folded tertiary structure of the Ade−Apt-A complex triggered the release of the labeled nucleic acids from the protein, leading to a strong decrease in the fluorescence anisotropy. The key factors involved in the fluorescence anisotropy change were considered through the development of a competitive displacement model, and the optimal tracer candidate was selected for the Ade assay under buffer and realistic (diluted human serum) conditions. The SSB-assisted principle was found to operate also with another aptamer system, i.e., the antiargininamide DNA aptamer, and a different biosensing configuration, i.e., the sandwich-like design, suggesting the broad usefulness of the present approach. This sensing platform allowed generation of a fluorescence anisotropy signal for aptamer probes which did not operate under the direct format and greatly improved the assay response relative to that of the most previously reported small target FPAA.

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

“…16C-TMR-A25 and 23A-TMR-A25 can be used as a pair of aptamer probes for FA sensing, showing complementary FA response to ATP. The obtained sensitivity for ATP analysis in this work is comparable to that obtained in many aptamer-based FA assays and fluorescence assays for ATP (Cui et al, 2012;Feng et al, 2014;Juskowiak, 2011;Liu et al, 2009Liu et al, , 2013Sassolas et al, 2011;Zhu et al, 2012).…”

“…proteins and oligonucleotides) or nanomaterials (e.g. 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.…”

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

“…31−34 The comparison in assay performance with other FA assays for OTA and aptamer-based FA assays for small molecules is summarized in Table S4 in the Supporting Information. [25][26][27][28][29][31][32][33][34]57,58 ■ CONCLUSION In summary, we demonstrated a simple fluorescence anisotropy strategy for direct analysis of a small molecule, OTA, with a TMR-labeled anti-OTA aptamer. It relied on the changes of the intramolecular interaction between TMR and guanine bases of aptamer and the subsequent local rotation of TMR upon target binding.…”

“…These results show the fluorophores labeled on the same site of the aptamer display distinct behaviors in response to OTA binding, significantly depending on the photophysical properties and molecular structures of the fluorophores. 20,26,32,38,39,43 Clearly, TMR-labeled aptamer is preferred for OTA analysis as it can give more sensitive FA response to OTA. On the basis of the above investigation, T10-TMR-O36 was chosen as a favorable aptamer probe in FA analysis of OTA.…”

Fluorescent sensing ochratoxin A with single fluorophore-labeled aptamer

“…Usually, fluorescent dyes can be readily introduced on the ends of the aptamers through chemical synthesis to fabricate the aptamer switch [5][6][7][9][10][11][12][13][14]. It is also feasible to conjugate fluorophores on the internal bases or to use the fluorescent analogue of the bases in oligonucleotides for sensing [5][6][7][15][16][17][18].…”