In Vitro Selection Of Aptamers To Inactivate Pseudomonas Aeruginosa By SELEX And Primary Application

【Objective】We have applied Systematic Evolution of Ligands by Exponential enrichment(SELEX) against the whole inactivate Pseudomonas aeruginosa ( P. aeruginosa)as a complex target to select single-stranded DNA-ligands (aptamers) from a 96nt single stranded DNA(ssDNA) random library,and establish a rapid diagnosis method by fluorophore-labed aptamers.【Methods】1. To obtain anti- inactivate P. aeruginosa aptamers with high affinity and specificity, the research started with the synthesis of a 96nt random oligonucleotides DNA library containing a central region of 60 random nucleotides flanked by a 5' and a 3' region of constant sequence. Then the ssDNA pool was subjected to 15 rounds of SELEX selection. We applied Enzyme-linked aptamer direct assay (ELADA) to determine the binding capabilities between the aptamers and P. aeruginosa every one round of selection according to Digoxigenin -anti-digoxigenin-AP system.2. The counterselection and un-counterselection of ssDNA binding to S. maltophilia and A. baumannii were performed after 12 rounds and 14 rounds, and the comparison of binding capabilities between them were also determined by ELADA.3. PCR products of the 6, 10 and 15 rounds of selection were cloned and sequenced. Relative softwares were employed to analyze the primary structure and prediction secondary structure of the aptamers.4. We isolated three of 24 DNA sequences that specifically bind to P. aeruginosa. and determined their affinity. We used ELADA and DFA to analyze the sensitivity and specificity of F23 clone aptamer binding to non-zymogenic.5. Establish a rapid diagnosis method by fluorescence labled aptamers: we applied 5'-FAM and 5'-TAMRA labed aptamer F23 to directly bind non-zymogenic, wash and mount, then analyzed by fluorescence microsopy.【Results】1. The results showed that a significant signal of binding capabilities over background and data ranged from 0.022 to 0.448 absorbance units following the selection increasing. The binding ssDNA aptamers were not increasing when the binding sites were full.2 There was signification deviation between the counterselection and un-counterselection process. The minimal ratio of binding capabilities was 1.5 times in the 15 rounds of selection, the max was 53 times in 14 rounds of selection.3. In order to assess our protocol, the enrichment of aptamers that bind to P. aeruginosa were monitored after 6, 10 and 15 rounds of selection. Multiple sequence alignments using ClustaX and Meg2 revealed that no enrichment was evident after 6 and 10 rounds of selection and 15 un-counterselection; however, after 15 couterselection, 24 cloned bacteria were sequenced and classified 10 families which had conserved sequences except the 10th.2 of 24 DNA sequences were modestly enriched which were F23 and F47, and the percentage of identity was reached 97%.4. We used an enzyme-linked aptamer direct assay (ELADA) to prioritize the aptamers from the P. aeruginosa selection for further characterization. This assay provided a rapid assessment of the relative binding capabilities of many aptamers from selection experiment .The results suggested that aptamer F23, F47 and F17 had the higher affinity for P. aeruginosa, the equilibrium dissociation constant (Kd) of 14.55, 77.46 and 493.3nM. Thus, we chose the aptamer F23 for further characterization.The sensitivity and detection of the target is attained as low as 2×106 bacteria. When tested in ELADA and DFA, the aptamer F23 exhibited specificity in its ability to bind only to P. aeruginosa but not to S. maltophilia and A. baumannii.5 We applied the 5'-FAM and 5'-TAMRA labed aptamer F23 to directly bind non-zymogenic, the result was the fluorescence intensity of binding to P. aeruginosa was much stronger than the other bacteria. Especially 5'-TAMRA labed aptamer was able to decrease the interference of the non-specificity background of tissues spontaneous fluorescence.【Conclusions】We have applied Systematic Evolution of Ligands by Exponential enrichment(SELEX) against the whole inactivate Pseudomonas aeruginosa(P. aeruginosa)and performed counterselection of ssDNA binding to other Pseudomonas to select single-stranded DNA-ligands(aptamers) from a single stranded DNA(ssDNA) random library after 15 rounds of selection. The selected aptamers were analyzed by ELADA and DFA.We has successfully isolated a set of 24 unique DNA sequences that specifically bind to P. aeruginosa from the random library. The research was the foundation of establishing a quick diagnostic method to detect P. aeruginosa with fluorophore-labed aptamers.