In vitro Selection
and Affinity Function of the Aptamers to Bacillus anthracis Spores by
SELEX
ZHEN Bei1, SONG
Ya-Jun, GUO Zhao-Biao, WANG Jin, ZHANG Min-Li, YU Shou-Yi1, YANG
Rui-Fu*
( Institute of Microbiology and Epidemiology, the Academy of Military Medical
Sciences, Beijing 100071, China；
Department of Epidemiology, the First Military Medical University, Guangzhou
510515, China )
Abstract To obtain
oligonucleotide aptamers, specifically binding to Bacillus anthracis
spores, and to find the relationship between the structures and the affinities,
and to determine whether the aptamers can be used as a novel molecule for spore
detection, a synthetic 35 mer random DNA library was subjected to 18 rounds of
selection by using SELEX (systematic evolution of ligands by exponential
enrichment) protocol against spores of Bacillus anthracis
vaccine strain A. 16R. The selected aptamers were cloned and sequenced.
Software packages CLUSTALX (1.8) and DNASIS v2.5 were employed to analyze the
conserved sequences and second structures of the aptamers, respectively.
Affinities of aptamers to the spores were visualized by biotinstreptavidinhorseradish
peroxidase system. DAB was used to visualize signals, as an assay method. A
membrane-based hybrid sandwich assay was developed for detecting Bacillus
anthracic spores by using a 5′-biotinylated ssDNA aptamers and
anti-spore antibodies. PCR amplification band pattern of the first round
selection was different from that of the ninth round. The binding assay
demonstrated that the affinity of the eighteenth round pool increased
thirty-seven folds more than that of the first round pool. The affinities of
the aptamers were different: the highest A at 450 nm was 1.20,
and the lowest was 0.20. The secondary structure analysis revealed possible
stem-loop and hairpin structures for binding to the spores. The colorimetry on
the immuno-membrane got the best signal with a ratio of 16 &mgr;g aptamer
to 4×107 spores. A set of aptamers with considerable binding
affinity to Bacillus anthracis spores was successfully
selected from the initial random ssDNA pool. The stem-loop and hairpin at 5′
end of the aptamers worked as the main motif in the interaction between
oligonucleotides and spores, while the neighbor bases of the triple structure
might affect the stability. Therefore ssDNA aptamers seem to be a type of
potential diagnostic molecule.
Key words Bacillus anthracis spores; aptamer;
affinity; immuno membrane
*Corresponding author：Tel, 86-10-83820748; Fax,
86-10-66948595；e-mail,[email protected]