In Vitro Selection Of Aptamers To SpA By SELEX And Primary Application

[Objective]To obtain aptamers which have property of high specificity and affinity against SpA,SpA molecules,which have bound to magnetic beads, serve as targets and screen out theaptamers from synthesized96nt random oligonucleotide library through systematic evolutionof ligands by exponential enrichment (SELEX) technology. Meanwhile, we resort to establishan ELISA method for detecting SpA with aptamers labeled with biotin and find out theaptamers which can block the SPA binding sites, eventually achieve the aim of controllingStaphylococcus aureus infection after applying with them.[Methods]1. We packaged Dynal magnetic bead with SPA, and then detected the packaging efficiencythrough BCA assay. Subsequently, we detected whether the structure of SPA changed afterpackaging through immunoabsorb assay.2. In vitro, we constructed an oligonucleotide library which has a length of96nt base in theoligonucleotide, which comprise a fixed sequence at both ends of oligonucleotide and60nt ofrandom sequence in the middle. And then acquired the aptamers which have property of highspecificity and affinity to SPA by increasing the intensity of screening (increasing tRNAamount, shorting the time of integration and increasing the frequency of vibration, etc.).3. Counter selection: Before the cycle of7,9and12screening, weremoved SpA non-specificbinding of ssDNA by combining ssDNA library with blank beads.Then, the remained ssDNA library which did not combining with the blank beads was asscreening library screening beads combine with SpA.4. To observe ssDNA enrichment situation, we have calculated the ratio of the amount ofssDNA combining with SpA to the total input library of2,4,6,7,9,10,11,12th round bydetermination of the fluorescence value of the total input library and unbound ssDNA withssDNA screening library Labeled by fluorescence Group.5. PCR products of the12th rounds of selection were cloned and sequenced. Relativesoftwares were employed to analyze the primary structure and prediction secondary structureof the aptamers.6. Determine the binding capability of aptamer family to SPA by using Dot-blot.7. Established an ELISA method for detecting SPA and a standard curve with aptamer toSpA.8. Established a method of IgG binding to beads coated with SPA, and then observe theinhibitive effect of aptamer between SPA and IgG.9. From the view of Cytology, Observe the inhibitive effect of aptamer in defendingStaphylococcus aureus and SPA based on the secretion of IL-8and phosphorylation of P38.10. In vivo, observed the Staphylococcus aureus infection in a mouse pneumonia, bacteremiaand death under the role of SPA aptamer, then observed the leukocyte recruiting in mouselungs after infection with Staphylococcus aureus or SPA. [Result]1. SPA efficiently coated beads and it can also effectively combine with IgG after packaging2. With the increase of screening cycles, the fluorescence value derived from SpA bindingto ssDNA enriched library increased. The binding rate of the12th cycle is45.6percent andthe first cycle is2.1percent, respectively. Once the binding sites between SpA and ssDNAenrichment library saturated, The binding rate s would not increase.3. We got sequencing result of45clones among48clones, and the analysis of obtainedsequence indicated the secondary structure of all sequences mainly have stem-loop or hairpinstructure. And then we selected five family for further analysis based on the sequence andstructure of clones, we found the homology between the various family mostly less than70%4. Among five family, the first, the second and the third family has the strongest capabilityof binding to SpA. Result of affinity determination showed the kd values of four family were:17.31±6.24nM;25.22±7.51nM,24.27±6.98nM and53.18±7.58nM, respectively. Andthe first, the second and third family can specifically bind to the SpA by using colloidalfiltration assay.5. We set up an ELISA method to detect SpA aptamer which were labeled with biotin andthis method can quantify the number of aptamer.6. Aptamer can significantly inhibit the binding of IgG to SpA and result in the amount ofIgG to SPA decrease from36.10%to6.4%.7. Staphylococcus aureus or interactions between SPA and TNFR in human epidermal cellspromote phosphorylation of P38, and the secretion of IL-8increased as a result. After addedthe first family of aptamers, the concertration of IL-8decreased from971.67±359.40pg/mlto487.08±189.09pg/ml under the role of bacteria, in addition, the concertration of IL-8decreased from778.08±278.52pg/ml to255.58±136.76pg/ml under the role of SPA.8. In mouse model with infection of Staphylococcus aureus, simultaneously added aptamersin mice,the incidence of pneumonia decreased from58.33%to16.67%,The incidence ofbacteremia decreased from58.33%to25%. The leukocyte recruitment Intrapulmonary havealso been improved, from58.08%±30.55%to29.83%±16.44%(Staphylococcus aureus),from34%±9%to16%±5%(SpA), respectively.【Conclusions】In this study, after12cycle of screening and counter selection to avoid non-specificbinding, we firstly obtained the ssDNA aptamer against SpA through systemic evolution ofligands by exponential enrichment (SELEX) technology. Following this, we use dot-blot todetect the combination of aptamer with SPA and establish an ELISA method to detect SpAaptamer. In addtion, present study screened out a specific aptamer against SpA anddemonstrated the blocking role of aptamer. Meanwhile, we also verified the capability ofaptamer against bacterial infection through inhibiting SPA, all of these results will lay thefoundation for finding new target site against Staphylococcus aureus in the future.