In Vitro Selection Of Aptamers To Psendomonas Aeruginosa And Primary Application

【Objective】To screem oligonucleotide aptamers , which bind to P. aeruginosa with high affinity and specificity by in vitro selection , to detect the binding activity of the aptamers and validate its binding rate and specificity against P. aeruginosa.【Methods】1. To obtain oligonucleotide aptamers which bind to P. aeruginosa with high affinity and specificity,the research started with the synthesis of a 78nt random oligonucleotides DNA library containing a central region of 35 random nucleotides flanked by a 5' and a 3' region of constant sequence. Then the ssDNA pool was subjected to 9 rounds of selection of binging-washing-separation–PCR amplifying by using SELEX method against P. aeruginosa. To increase specificity of ssDNA binding to P. aeruginosa,counterselection was performed with P.mendocina,P.stutzeri and P.alcaligenes every 1 round from round 5 respectively. Digoxigenin -anti-digoxigenin-AP system was applied to determine the binding activity between the aptamers and P. aeruginosa every 1 selection round.2. PCR products of the 9 of round selection were cloned and sequenced.Relative software was employed to analyze the primary structure and prediction secondary structure of the aptamers.3. The binding activity of fluorescein-labled aptamers against P. aeruginosa was determined by Mini-Fluorometer,then the highest one was chosen to bind with P. aeruginosa,P.mendocina,P.stutzeri and P.alcaligenes respectively to detect the binding specificity of the aptamer. 【Results】1. After 9 rounds of selection and amplification,the aptamers against P. aeruginosa were enriched gradually , an increasing tendency of binding activity between the aptamers and P. aeruginosa can be detected.Selected products in every 1 round were amplified through PCR with Dig-modified primers.The amplified products binding to the P. aeruginosa was visualized by Digoxigenin -anti-digoxigenin-AP system.The binding assay demonstrated that the binding activty of the ninth round pool increased 3 times from that of the first round pool.2. The aptamers pool obtained from 9 of round selection was cloned.27 clones from 200 clones were randomly selected to be sequenced.According to base number of the sequence,the 23 clones were divided into 2 groups.Group A consisted of 19 fragments,which were of the same size as the expected one.Group B consisted of fragments that had 1 or 3 basic radical less in random region than the expected one. Software packages were employed to analyze the conserved sequences and second structures of the aptamers .Stem-loop are the main motif in prediction secondary structure which may be the binding sites between the aptamers and target.There were 6 aptamer clones enjoy the absolute identical sequence motif,and there were 20 nucleotide bases of G in their random region,which might help to form the G-quartets.In addition,some aptamer clones had no significant sequence similarities to the rest. The secondary structure of some aptamer clones were resemble ,but there were few homogenous sequences among them,such as clone 2-26,2-61,2-119 and clone 2-24,2-46 and clone 2-12,2-135.3. Binding assay was taken to find the aptamers with higher binding activity.The binding activity of the aptamers were different.Clone 2-61 was the highest and its binding activity was 15.3%;Clone 2-18 was the lowest and its binding activity was5.6%.Then the clone 2-61 aptmer binded to P. aeruginosa, P.mendocina, P.stutzeri and P.alcaligenes at the same time,the binding activity were 16.6%, 3.8%, 2.6%, 4.5% respectively.【Conclusions】single-stranded DNA aptamers with capability to bind P. aeruginosa were selected by counter systematic evolution of ligands by exponential enrichment (SELEX) method in China.The binding activity of the aptamers obtained from this method were determined ,the sepuence of the aptamer targeting to P. aeruginosa with high binding activty and high specificity was validated.The research was the foundation of establishing a quick diagnostic method to detect P. aeruginosa with fluorophore-labed aptamers.