Identification of the Target Binding Site of Ethanolamine-Binding Aptamers and Its Exploitation for Ethanolamine Detection

Abstract

Aptamers are promising recognition elements for sensitive and specific detection of small molecules. We have previously selected ssDNA aptamers for ethanolamine, one of the smallest aptamer targets so far. The work presented here focuses on the determination of the binding region within the aptamer structure and its exploitation for the development of an aptamer-based assay for detection of ethanolamine. Sequence analysis of the aptamers resulted in the identification of a G-rich consensus sequence, which was able to fold in a typical two- or three-layered G-quartet structure. Experiments with stepwise truncated variants of the aptamers revealed that the consensus sequence is responsible and sufficient for binding to the target. On the basis of the knowledge of the aptamers binding site, we developed an aptamer-based microarray assay relying on competition between ethanolamine and an oligonucleotide complementary to the consensus sequence. Competitive binding of ethanolamine and fluorescently labeled complementary oligonucleotides resulted in fluorescence intensities dependent on ethanolamine concentration with a limit of detection of 10 pM. This method enables detection of small molecules without any labeling of analytes. The competitive assay could potentially be transferred to other aptamers and thus provides a promising system for aptamer-based detection of diverse small molecules.

Aptamer-based biosensor for label-free detection of ethanolamine by electrochemical impedance spectroscopy

“…After that, the ∆R CT changed slowly with the increased EA concentration from 160 nM to 1.6 mM, whereas upon decreasing the concentration of EA to 0.08 nM, no distinct ∆R CT change was observed compared to that in buffer solution, which was due to the less G-quadruplex/EA complexs formed on the electrode films. And the detection limit was determined to be 0.08 nM, which is comparable to the fluorescent assay for EA (0.01 nM) [11] and much lower than that of chromatographic methods as shown in Table S1. Furthermore, we also investigated the stablity of the Ap-DNA film.…”

“…On the basis of the findings, Heilkenbrinker group reported an aptamer-based microarray assay based on competition between EA and the fluorescein-labeled DNA strand, which is complementary to the aptamer DNA sequence immobilized on the microarray surface [11]. This methods made great improvements in detection techniques for EA.…”

“…This can be rationalized that the formed G-quadruplex on the electrode surface can provide binding sites for EA, forming EA/G-quadruplex complex [11], which hinders the electron transfer [36], resulting in an increased R CT .…”

Design and evaluation of split-ring resonators for aptamer-based biosensors

“…In a first step, the aptamers' ability to selectively bind CRP is tested. Therefore, 5 amino-modified aptamers are spotted on a 3-D aldehyde-modified microarray slide (PolyAn, Berlin, Germany), similar to the procedure described in Heilkenbrinker et al (2015), and subsequently exposed to solutions with different concentrations of fluorescence-labeled CRP to perform quantitative measurements. Furthermore, the aptamers are exposed to solutions with different concentrations of fluorescence-labeled bovine serum albumin (BSA) to test the selectivity of the aptamers.…”

Porous Silicon Biosensors Employing Emerging Capture Probes

“…Thus, in the presence of the target, the target will replace the complementary oligonucleotide, while the release of the oligonucleotide can be detected and quantified via different labeling techniques. This strategy has been already successfully applied for the detection of ethanolamine, which is the smallest analyte against which an aptamer has been selected [66]. In more sophisticated sensing schemes, the aptamer can simultaneously act as both the bioreceptor and the transducer.…”