[In vitro selection and affinity function of the aptamers to Bacillus anthracis spores by SELEX]

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 biotin streptavidin horseradish 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 microgram aptamer to 4x10(7) 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.