Selection And Application Of Aptamer To Acinetobacter Baumannii

【Objective】We have applied SELEX （Systematic Evolution of Ligands by Exponentialenrichment）against the whole Acinetobacter Baumannii(A.Baumannii) as a complextarget to select single-stranded aptamers from a88nt single stranded DNA(ssDNA)random library.With epoxyethaneacrylic beads as screening medium, and the use ofdigoxinbiotin-alkaline phosphatase show color system, we tested and proved the highaffinity of aptmer to Acinetobacter Baumannii【Methods】1. Synthetize a88nt random oligonucleotides DNA library in vitro,which containsa central region of42random nucleotides flanked by a5’ and a3’ region of constantsequence. After15rounds of SELEX selection, we have screened aptamers toAcinetobacter Baumannii with high affinity and specificity. We applied Biotin-anti-digoxigenin-AP system to test the binding capabilities between the aptamersand Acinetobacter Baumannii.3. We perform the counterselection of ssDNA binding to Acinetobacter Los Philipinesand Acinetobacter Hemolysis after12th round, and compare the binding capabilitiesusing the Biotin-anti-digoxigenin-AP system.4. We have cloned the PCR products of the9th,12th and15th rounds ofselection.And related softwares were employed to analyze the primary structureand secondary structure of the aptamers.5. Three of42DNA sequences which specifically bind to A. Baumannii and wereisolated and affinity were also determined. ELADA and DFA were used to analyzethe sensitivity and specificity of B20aptamer.【Results】1. With the increase of the number of screening round,the OD value of binding theAcinetobacter Baumannii and ssDNA was also on the rise ranging from0.023to0.469. The binding ssDNA aptamers were not increasing when the binding sites were full. when ssDNA library and Acinetobacter Baumannii binding sites tendsto be gradually saturated, adsorption of ssDNA will no longer increase.2In the11th round, O.D value is lower, this may be due to the selection pressure(choose/evolutionary pressure) influence, the expected nucleotide have enhancednonspecifically..3. Comparing the combination of OD value with and without the counterselection, wehave found obvious difference.The figure shows the lowest ratio of15rounds isonly1.7times, and the highest ratio of the12th round is40times..4Cloning sequencing results show that there is no sequence enrichment in9th,12th,15th round without conterselection.With sequencing aptmer from15throunds,we have found that in23expected sequences, three sequences haveenriched and they have high similarity of92%(B20, B35and B44).Using DNASTAR software to compare the primary structure, these sequences can be dividedinto nine Family (Family).And then we use DNASISV2.5software to calculaterespectively. Some sequences were not related to any of those famlies.6. Combination of the OD value of cloning aptmer to Acinetobacter Baumannii B20,B35and B44were higher than others. With the affinity determination, we haveknown that B201Kd is7.89nM, B35Kd is86.83nM, the B44Kd is103.99nM.With sensitivity determination for B20, lowest content of bacteria can be2.5x106【Conclusions】We screened the bacterium of other acinetobacter with SELEX.After15rounds, wehave obtained ssDNA aptmer to Acinetobacter Baumannii.UsingBiotin-anti-digoxigenin-AP system and DFA technology to analyze thespecificity,sensitivity, and affinity,we have successfully sreened the aptmer toAcinetobacter Baumannii with high affinity and strong specificity...