1 research outputs found
Identification of the Target Binding Site of Ethanolamine-Binding Aptamers and Its Exploitation for Ethanolamine Detection
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