Details
| Original language | English |
|---|---|
| Pages (from-to) | 677-685 |
| Number of pages | 9 |
| Journal | Analytical chemistry |
| Volume | 87 |
| Issue number | 1 |
| Publication status | Published - 10 Dec 2014 |
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.
ASJC Scopus subject areas
- Chemistry(all)
- Analytical Chemistry
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In: Analytical chemistry, Vol. 87, No. 1, 10.12.2014, p. 677-685.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Identification of the Target Binding Site of Ethanolamine-Binding Aptamers and Its Exploitation for Ethanolamine Detection
AU - Heilkenbrinker, Alexandra
AU - Reinemann, Christine
AU - Stoltenburg, Regina
AU - Walter, Johanna-Gabriela
AU - Jochums, André
AU - Stahl, Frank
AU - Zimmermann, Stefan
AU - Strehlitz, Beate
AU - Scheper, Thomas
PY - 2014/12/10
Y1 - 2014/12/10
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84920407832&partnerID=8YFLogxK
U2 - 10.1021/ac5034819
DO - 10.1021/ac5034819
M3 - Article
C2 - 25435319
AN - SCOPUS:84920407832
VL - 87
SP - 677
EP - 685
JO - Analytical chemistry
JF - Analytical chemistry
SN - 0003-2700
IS - 1
ER -