Screening Of SsDNA Aptamers Specific To Staphylococcus Aureus By SELEX And Their Preliminary Application

SELEX (Systematic Evolution of ligands by Exponential Enrichment) is a combinatorial chemistry technology for structural, functional and molecular evolutional study of nucleic acid. The aptamers derived from this technology have promising application in basic research, drug screening, and diagnosis. The objective of our research is to establish a whole bacterium–based subtractive selection method with random ssDNA library targeting Staphylococcus aureus (S. aureus) and to gain a panel of specific ssDNA aptamers, which will be of potential use for the rapid and sensitive determination of S. aureus in the future.Before the SELEX selection, a novel method for ssDNA preparation was set up by PCR with a stem-loop reverse primer. Then subtractive SELEX selection against S. aureus with a 88 base ssDNA library containing 45 bases of random sequence was done, followed by counter selection to Streptolococcus and Staphylococcus epidermidis. After seven rounds of bacterium-based selection, the enrichment of each round was tested by isotope labeled ssDNA binding assay. Results showed that the fifth round pool was of the best enrichment. Then the fifth round pool was cloned and sequenced. The secondary structure of the sequences was analyzed by RNA STRUCTURE and MEME online software. Eleven sequences were picked out for binding assay by confocal imagining and flow cytometry analysis, and five of them showed high specificity and affinity to S. aureus with Kd on nanomolar leve. Futhermore, The binding assay also indicated that the five aptamers as a panel of probes had much better effect than a single aptamer in the recognization of S. aureus. The results of our work provided an important information that a set of specific aptamers can be used to identify pathogenic microorganism rapidly in the future instead of one single aptamer.