体外筛选铜绿假单胞菌适体的研究及初步应用
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摘要
【目的】
体外筛选出高亲和力高特异性针对铜绿假单胞菌的适体群;测定适体与铜绿假单胞菌的结合活性,确定其结合率及特异性。
【方法】
1.在体外构建两端为固定序列,中间为35个碱基随机区,全长78个碱基的寡核苷酸库,以纯培养的铜绿假单胞菌为靶标,将二者混合,经过九轮的吸附-漂洗-洗脱-扩增的SELEX过程,筛选针对铜绿假单胞菌高亲和力高特异性的适体,并从第五轮起每隔一轮分别用荧光假单胞、斯氏假单胞和产碱假单胞菌进行反筛,以提高筛选的特异性。每隔一轮利用地高辛-抗地高辛抗体-碱性磷酸酶系统测定ssDNA富集库与铜绿假单胞菌的结合率。
2.将第九轮的筛选产物——寡核苷酸片断进行克隆、测序并用相关软件分析其一级和二级结构。
3.用微量荧光分光光度计检测荧光素标记适体与铜绿假单胞菌的结合率,在5个测序克隆中选出结合率最高的一条适体,分别与铜绿假单胞、门多萨假单胞、斯氏假单胞和产碱假单胞菌做结合反应,以初步检测其特异性。
【结果】
1.经过九轮的筛选和扩增,使得针对铜绿假单胞菌的适体逐步得到富集,每轮的ssDNA文库与铜绿假单胞菌的结合率呈上升趋势。将每轮的筛选产物用标有地高辛的引物扩增后,与铜绿假单胞菌进行结合实验,地高辛-抗地高辛抗体-碱性磷酸酶显色系统提示,第九轮筛选获得的富集库与铜绿假单胞菌结合后显色的OD值较第一轮的OD值提高将近3.2倍。
2.在200个克隆子中随机选取27个克隆进行了测序,将测序结果按碱基多少分成A、B两群,A群19条与预期片段大小一致;B群为中间随机区少于35个碱基的片段。将测序适体以计算机软件分析其保守序列及二级结构。二级结构预测以茎-环结构为主,这可能是适体与靶标作用的结构基础。其中适体序列完全相同的共有6条,此序列随机区富含20个G,可能为G四聚体的形成基础。此外,有些序列是单个序列,与任何家族无同源。某些一级结构无同源性的适体克隆二级结构模拟却极为相似,如2-26、2-61、2-119号克隆,2-24、2-46号克隆,2-12、2-135克隆序列等。
3.结合实验显示,2-61号克隆适体与铜绿假单胞菌的结合率最高,为15.3%,2-106号克隆适体与铜绿假单胞菌的结合率最低,为5.6%;2-61号克隆适体与铜绿假单胞、斯氏假单胞菌、产碱假单胞菌和门多萨假单胞,进行结合反应后,测得F值分别为16.6%、3.8%、2.6%和4.5%。
【结论】
本研究在国内首次利用反向-指数富集配体系统进化(SELEX)技术筛选出针对铜绿假单胞菌的ssDNA适体,并对筛选出的适体进行了结合率及特异性的检测,初步确定了结合率较高特异性较好的适体分子,为荧光基团标记的适体快速鉴定铜绿假单胞菌检测方法的建立奠定了基础。
【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.
体外筛选出高亲和力高特异性针对铜绿假单胞菌的适体群;测定适体与铜绿假单胞菌的结合活性,确定其结合率及特异性。
【方法】
1.在体外构建两端为固定序列,中间为35个碱基随机区,全长78个碱基的寡核苷酸库,以纯培养的铜绿假单胞菌为靶标,将二者混合,经过九轮的吸附-漂洗-洗脱-扩增的SELEX过程,筛选针对铜绿假单胞菌高亲和力高特异性的适体,并从第五轮起每隔一轮分别用荧光假单胞、斯氏假单胞和产碱假单胞菌进行反筛,以提高筛选的特异性。每隔一轮利用地高辛-抗地高辛抗体-碱性磷酸酶系统测定ssDNA富集库与铜绿假单胞菌的结合率。
2.将第九轮的筛选产物——寡核苷酸片断进行克隆、测序并用相关软件分析其一级和二级结构。
3.用微量荧光分光光度计检测荧光素标记适体与铜绿假单胞菌的结合率,在5个测序克隆中选出结合率最高的一条适体,分别与铜绿假单胞、门多萨假单胞、斯氏假单胞和产碱假单胞菌做结合反应,以初步检测其特异性。
【结果】
1.经过九轮的筛选和扩增,使得针对铜绿假单胞菌的适体逐步得到富集,每轮的ssDNA文库与铜绿假单胞菌的结合率呈上升趋势。将每轮的筛选产物用标有地高辛的引物扩增后,与铜绿假单胞菌进行结合实验,地高辛-抗地高辛抗体-碱性磷酸酶显色系统提示,第九轮筛选获得的富集库与铜绿假单胞菌结合后显色的OD值较第一轮的OD值提高将近3.2倍。
2.在200个克隆子中随机选取27个克隆进行了测序,将测序结果按碱基多少分成A、B两群,A群19条与预期片段大小一致;B群为中间随机区少于35个碱基的片段。将测序适体以计算机软件分析其保守序列及二级结构。二级结构预测以茎-环结构为主,这可能是适体与靶标作用的结构基础。其中适体序列完全相同的共有6条,此序列随机区富含20个G,可能为G四聚体的形成基础。此外,有些序列是单个序列,与任何家族无同源。某些一级结构无同源性的适体克隆二级结构模拟却极为相似,如2-26、2-61、2-119号克隆,2-24、2-46号克隆,2-12、2-135克隆序列等。
3.结合实验显示,2-61号克隆适体与铜绿假单胞菌的结合率最高,为15.3%,2-106号克隆适体与铜绿假单胞菌的结合率最低,为5.6%;2-61号克隆适体与铜绿假单胞、斯氏假单胞菌、产碱假单胞菌和门多萨假单胞,进行结合反应后,测得F值分别为16.6%、3.8%、2.6%和4.5%。
【结论】
本研究在国内首次利用反向-指数富集配体系统进化(SELEX)技术筛选出针对铜绿假单胞菌的ssDNA适体,并对筛选出的适体进行了结合率及特异性的检测,初步确定了结合率较高特异性较好的适体分子,为荧光基团标记的适体快速鉴定铜绿假单胞菌检测方法的建立奠定了基础。
【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.
引文
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[5] McCauley T G,Hamaguchi N,Stanton M.Aptamer-based biosensor arrays for detection and quantification of biological macromolecules. Anal Biochem, 2003, 319(2): 244-250
[6] Jhaveri S, Rajendran M, Ellington AD . In vitro selection of signaling aptamers. Nat Biotechnol,2000,18(12): 1293-1297
[7] Yamana K, Ohtani Y, Nakano H,Saito I. Bis-pyrene labeled DNA aptamer as an intelligent fluorescent biosensor. Bioorg Med Chem Lett,2003,13(20): 3429-3431
[8] Zhang P, Beck T, Tan W. Design of a Molecular Beacon DNA Probe with Two Fluorophores . Angew Chem Int Ed Engl, 2001,40(2), 402-405
[9] Li J J, Fang X, Tan W. Molecular aptamer beacons for real-time protein recognition. Biochem Biophys Res Commun,2002,292(1) 31-40
[10] Yamamoto R , Baba T , Kumar P K. Molecular beacon aptamer fluoresces in the presence of Tat protein of HIV-1. Genes Cells,2000, 5(5): 389-396
[11] Nutiu R, Li Y. Structure-switching signaling aptamers. J Am Chem Soc,2003,125(16): 4771-4778
[12] Nutiu R,Li Y. In Vitro selection of structure-switching signaling aptamers. Angew Chem Int Ed Engl,2005, 44(7):1061-1065
[13] Nutiu R,Li Y.Aptamer with fluorescence-singaling properties.Methods,2005,37(1): 16-25
[14] Merino E J, . Weeks K M. Fluorogenic resolution of ligand binding by a nucleic acid aptamer.J Am Chem Soc,2003,125(41) :12370-123771
[15] Stojanovic MN, Kolpashchikov DM. Modular aptameric sensors. J Am Chem Soc,2004, 126(30):9266–9270
[16] Stojanovic MN, Landry DW. Aptamer-based colorimetric probe for cocaine. J Am Chem Soc,2002,124(33): 9678–9679
[17] Tombelli S,Minunni M,Mascini M.Analytical application of aptamers .Biosens Bioelectron,2005,20(12):2424-2434
[18] Jiang Y, Fang X, Bai C. Signaling aptamer/protein binding by a molecular light switch complex. Anal Chem,2004,76(17):5230–5235
[19] Zhou C, Jiang Y, Hou S, Ma B, Fang X, Li M. Detection of oncoprotein platelet-derived growth factor using a fluorescent signaling complex of an aptamer and TOTO. Anal Bioanal Chem,2006,384(5):1175-1180
[20] Ho HA, Leclerc M. Optical sensors based on hybrid aptamer/conjugated polymer complexes. J Am Chem Soc,2004,126(5):1384-1387
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[1] Cox JC, Rajendran M, Riedel T, Davidson EA, Sooter LJ, Bayer TS,Schmitz-Brown M, Ellington AD. Automated acquisition of aptamer sequences. Comb Chem High Throughput Screen, 2002,5(4):289-299
[2] Bock C, Coleman M, Collins B, Davis J, Foulds G, Gold L, Greef C,Heil J, Heilig JS, Hicke B. et al. Photoaptamer arrays applied to multiplexed proteomic analysis. Proteomics, 2004,4(3):609-618
[3] Eulberg D, Buchner K, Maasch C, Klussmann S. Development of an automated in vitro selection protocol to obtain RNA-based aptamers: identification of a biostable substance P antagonist. Nucleic Acids Res ,2005, 33(4):e45
[4] Nutiu R, Li Y.Structure-switching signaling aptamers:transducing molecular recognition into fluorescence singaling.Chemistry,2004,10:1868-1876
[5] McCauley T G,Hamaguchi N,Stanton M.Aptamer-based biosensor arrays for detection and quantification of biological macromolecules. Anal Biochem, 2003, 319(2): 244-250
[6] Jhaveri S, Rajendran M, Ellington AD . In vitro selection of signaling aptamers. Nat Biotechnol,2000,18(12): 1293-1297
[7] Yamana K, Ohtani Y, Nakano H,Saito I. Bis-pyrene labeled DNA aptamer as an intelligent fluorescent biosensor. Bioorg Med Chem Lett,2003,13(20): 3429-3431
[8] Zhang P, Beck T, Tan W. Design of a Molecular Beacon DNA Probe with Two Fluorophores . Angew Chem Int Ed Engl, 2001,40(2), 402-405
[9] Li J J, Fang X, Tan W. Molecular aptamer beacons for real-time protein recognition. Biochem Biophys Res Commun,2002,292(1) 31-40
[10] Yamamoto R , Baba T , Kumar P K. Molecular beacon aptamer fluoresces in the presence of Tat protein of HIV-1. Genes Cells,2000, 5(5): 389-396
[11] Nutiu R, Li Y. Structure-switching signaling aptamers. J Am Chem Soc,2003,125(16): 4771-4778
[12] Nutiu R,Li Y. In Vitro selection of structure-switching signaling aptamers. Angew Chem Int Ed Engl,2005, 44(7):1061-1065
[13] Nutiu R,Li Y.Aptamer with fluorescence-singaling properties.Methods,2005,37(1): 16-25
[14] Merino E J, . Weeks K M. Fluorogenic resolution of ligand binding by a nucleic acid aptamer.J Am Chem Soc,2003,125(41) :12370-123771
[15] Stojanovic MN, Kolpashchikov DM. Modular aptameric sensors. J Am Chem Soc,2004, 126(30):9266–9270
[16] Stojanovic MN, Landry DW. Aptamer-based colorimetric probe for cocaine. J Am Chem Soc,2002,124(33): 9678–9679
[17] Tombelli S,Minunni M,Mascini M.Analytical application of aptamers .Biosens Bioelectron,2005,20(12):2424-2434
[18] Jiang Y, Fang X, Bai C. Signaling aptamer/protein binding by a molecular light switch complex. Anal Chem,2004,76(17):5230–5235
[19] Zhou C, Jiang Y, Hou S, Ma B, Fang X, Li M. Detection of oncoprotein platelet-derived growth factor using a fluorescent signaling complex of an aptamer and TOTO. Anal Bioanal Chem,2006,384(5):1175-1180
[20] Ho HA, Leclerc M. Optical sensors based on hybrid aptamer/conjugated polymer complexes. J Am Chem Soc,2004,126(5):1384-1387