rHuEPO的特异性寡核苷酸适配体的筛选、评价及其应用
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摘要
促红细胞生成素(Erythropoietin, EPO)是一类重要的造血生长因子,主要由肾脏分泌产生。具有促进红细胞增殖和分化,提高红细胞运输氧的功能。近年随着基因工程等生物技术的迅速发展,人们可以在哺乳动物细胞株中实现EPO基因的转染,由此可获得大量具有生物活性的重组人促红细胞生成素(rHuEPO)。rHuEPO类制剂中的rHuEPO-α和rHuEPO-β在临床上广泛用于治疗肾性贫血和癌症化疗引起的贫血症状。
基于rHuEPO具有提高红细胞运输氧的药理功能,在耐力比赛中,存在运动员滥用此类药物以提高比赛成绩的可能性,因此世界反兴奋剂组织(WADA)的违禁药物清单中已明确将其列为肽类激素兴奋剂。目前开展的针对rHuEPO的检测方法,主要基于抗体作为传感或富集元件,以免疫分析或质谱分析为手段实现rHuEPO的检测。但抗体自身具有批间差异性、免疫原性和热不稳定性的局限性,将会阻碍目前基于抗体的免疫分析方法的特异性和灵敏度的提高。因此,针对抗体外新识别元件的研制,将对rHuEPO的分析检测提供更加有效的新思路和新途径。
适配体是一类可折叠形成一定三维结构用于特异性识别靶分子的单链DNA,RNA或修饰后的核酸分子,与抗体有类似的识别特性,但较抗体相比特异性更强,并具有无免疫原性、易从体外化学途径制备、及热稳定性更强的优点,这就为开发rHuEPO-α的高亲和力、高特异性适配体提供了新思路。因此实验设计采用凝集素导向的指数富集配基的系统进化(SELEX)技术,从全长80个碱基的单链脱氧寡核苷酸文库中,经8轮反复富集,成功筛选获得特异性识别rHuEPO-α的寡核苷酸适配体。并对筛选得到的部分高亲和力、高特异性的适配体进行结构分析。最终,基于具有天然茎环式二级结构的适配体807-39 nt,建立了一种新型的适配体型分子信标检测策略,实现rHuEPO-α的高灵敏度高特异性的检测。
本论文共分为四章。第一章为文献综述,从促红细胞生成素的功能／用途及研究概况、SELEX筛选技术的发展、分子信标的原理及设计策略三方面进行概括,引用文献115篇。
第二章详细介绍了rHuEPO-α的特异性寡核苷酸适配体的筛选过程。实验中采用凝集素导向的亲和树脂SELEX技术,将rHuEPO-α的唾液酸糖基部分固定后,实现针对其氨基酸序列的筛选,经N-乙酰葡萄糖胺特异性洗脱寡核苷酸与蛋白的复合物,经过8轮筛选后各轮(收敛)寡核苷酸文库的富集效率由13.8%提高到64.0%,同时各轮文库对于rHuEPO-α的选择性也由1.4增加到10.6。随后,对于第8轮富集文库的单克隆序列进行了序列比对分析,主要可以分为四个家族。Mfold模拟所得序列的二级结构主要呈现三大特征：紧密的茎环结构、多分支的茎环结构、少或无茎环结构。其中部分单克隆适配体的解离常数在几十nM到几百nM之间,亲和力最高的为适配体807(Kd=82±32 nM)。
第三章为rHuEPO-α的特异性寡核苷酸适配体的截短序列研究。对于具有相对较低的吉布斯自由能和较低解离常数的适配体807和813,进行了进一步序列截短分析研究,以优化和寻求最短的有效结合序列长度。在第8轮文库中所占比例最高的序列807,其完全随机区807-39nt具有紧密的茎环结构,解离常数低于全长序列807,说明807-39nt的茎环结构对于复合物的形成有着重要作用。序列813的最短有效截短长度为813-42nt,其3’引物端的部分序列参与了随机区的茎部配对,并有利于复合物的形成。
适配体807-39nt具有发卡式二级结构,主要由一富含G碱基的环部和长度为5对碱基的茎部组成。对于其茎部／环部的系列长度改变／碱基突变等研究表明,其环部为与靶分子rHuEPO-α相互作用的主要结合位点,而茎部的主要作用为稳定复合物的形成。并进一步针对靶蛋白的结合位点进行研究,去糖基化实验结果表明,当糖蛋白rHuEPO-α去除N连接的末端糖基后,仍可以与适配体807-39nt发生特异性的相互作用。因此,可初步推测,对于适配体807-39nt,蛋白rHuEPO-α与其作用的主要结合部位为其氨基酸序列。适配体807-39nt可以识别商品化注射液(益比奥和罗可曼)中不同类型的rHuEPO,亦从另一方面说明了rHuEPO的氨基酸序列为适配体807-39nt的主要作用区域。同时膀胱正常上皮组织的免疫组化实验证实适配体807-39nt特异性优于rHuEPO-α的单克隆抗体AE7A5,并发现适配体807-39nt与膀胱癌上皮组织间具有特异性相互作用。
适配体807-39nt具有天然的茎环结构,在第四章中,基于此点我们建立了特异性识别rHuEPO-α的适配体型分子信标检测策略。分别构建"Signal-on"和"Signal-off"两种分子信标模式,依据适配体荧光信号的变化判断其结合靶分子rHuEPO-α前后分子构象的改变。对于"Signal-on"体系,淬灭DNA序列的长度、稳定DNA的杂交位置和金属离子Mg2+的浓度是其获得高灵敏度的关键因素,在优化后的条件下,未经任何富集手段可实现1 nM rHuEPO-α的直接快速灵敏检测,其LOD为0.4 nM。
综上所述,本论文成功建立了凝集素导向的亲和SELEX技术,用于筛选rHuEPO-α的高亲和力、高特异性的ssDNA适配体。对筛选得到的适配体807和813进行了结构功能研究,并初步判定了其和rHuEPO-α的主要作用位点。其中亲和力和特异性最佳的适配体807-39nt(Kd=39±27 nM)有望成为新的传感元件,可为拓宽现有的rHuEPO-α检测方法,开发有效的针对rHuEPO-α诊断试剂提供了除抗体之外的新途径。
Erythropoietin (EPO) is a kind of significant occurring hematopoietic growth factor and produced mainly in the kidney, which has the ability of promoting the proliferation and differentiation of erythroid progenitor cells and enriches the count of oxygen-carrying red blood cells. In recent years, the technique of transfection of EPO gene in mammalian cell lines makes large scale production of biologically active recombinant human EPO (rHuEPO) convenient with the development of genetic engineering. In all kinds of EPO preparations, rHuEPO-αand rHuEPO-β, have been extensively used in clinic to treat for anemia associated with renal disease and cancer.
It is possible for rHuEPO-αto be abused in endurance sports to improve the performance by many athletes for its pharmacological action of enhancing the count of oxygen-carrying red blood cells. As a kind of well-known doping peptide hormone, erythropoiesis-stimulating agents are labeled clearly in the prohibited list of World Anti-Doping Code. Immunological or mass spectrometric-based techniques are mainly employed for its analysis with the aid of antibodies as a sensing and enrichment element. However, the limitation of anti-EPO antibody, such as batch-to-batch variability, immunogenicity and thermolability, will hinder the improvement of specificity and sensitivity in immunoassays. The effectively analytical strategies are greatly needed for detection of rHuEPO with the development of new recognition element.
Aptamers are single-stranded DNA, RNA, or modified nucleic acids that have the ability to form defined tertiary structures for specific target binding. They have the similar recognition characterization as antibody and have no immunogenicity, easy preparation by chemical synthesis in vitro and strong thermal stability, these unique merits of aptamer are providing a promising alternative way to pursue affinity ligands with high affinity and high specificity for rHuEPO-α. So our group adopted agglutinin-directed Systematic Evolution of Ligands by EXponential enrichment (SELEX) technique to generate aptamers for rHuEPO-α, and a series of aptamers with specifity for rHuEPO-a were successfully obtained from 80nt ssDNA library after 8 selection rounds. Some aptamers with high affinity and specificity were chosen to carry out structure analysis. Finally, a new pattern of aptameric molecular beacon strategy was developed for the detection of rHuEPO-a basd on aptamer 807-39nt with naturally stem-loop secondary structure.
The dissertation contains four parts, the first part is an overview on three aspects including instruction of function and purpose of EPO, development of SELEX technology and principle of molecular beacon. Total 115 references were cited.
In the second part, the selection procedure of aptamer with high affinity and specificity for rHuEPO-a were introduced in detail. The wheat germ agglutinin (WGA)-directed affinity chromatographic SELEX was adopted to bind the sialic acids moiety of rHuEPO-a, and exposing peptide chains for selection, then a high-concentrated N-acetylglucosamine solution was applied to elute the complex of rHuEPO-a and ssDNAs by the displacement of sialic acids moiety from WGA-Sepharose. After 8 rounds of selection, the enrichment efficiency of oligonucleotides was obviously improved from 13.8% to 64.0% by determination of the percentage of bound ssDNAs and the selectivity of ssDNAs for rHuEPO-a was increased from 1.4 to 10.6. The clones sequenced from the 8th pool were divided into four families, the characteristics of these secondary structure present tight hairpins, multi-branched hairpins and few or no hairpins. The dissociation constants of some sequences were nM level, the sequence 807 can bind with rHuEPO-a with the highest affinity and specificity (Kd=82±32 nM).
The third part is mainly on the truncation research of aptamers for rHuEPO-a. The sequences 807 and 813 with relative lower Gibbs' energy and nanomole dissociation constant were selected to carry out truncation research in order to optimize and find the effective binding motif. The random sequence 807-39nt with tight hairpin structure truncated from the most frequently repeated sequence 807 exhibited by far the highest binding for rHuEPO-a, which can account for the superior binding capacity of the hairpin structure. The aptamer 813-42nt was the shortest motif truncated from 813 for binding with rHuEPO-a, EMS A results indicate that the stem forming between the region sequence and partly 3'primer region might facilitate the binding capacity.
The secondary structure of 807-39nt is composed of a G-rich loop and a 5bp stem. A series of stem and loop variation experiments show that the recognition is occurred mainly in the loop part and the stem is functioned as a support to fix an enclosed loop in order to stabilize the complex of rHuEPO-α. Also the binding site of target rHuEPO-αwas further determined by the deglycosylation experiment, rHuEPO-αwithout N-glycosylation still remain the binding ability with 807-39nt. We might speculate that the binding site of protein is mainly the amino acid sequence, which is further confirmed by the binding between 807-39nt and Injection of Yi Bi Ao or Recormn. Also the results of immunohistochemistry experiments on normal urothelium showed that the specificity of aptamer 807-39nt was superior than anti-rHuEPO-αmAb AE7A5, and the aptamer 807-39nt has the ability to bind with malignant urothelium.
In the fourth part, we designed the aptameric molecular beacon (MB) for rHuEPO-αdetection, based on the stem-loop secondary structure of 807-39nt. Both "Signal-on" and "Signal-off' MB modes were developed, respectively, in which the conformational alteration of aptamer before and after binding to rHuEPO-a can be demonstrated in terms of the correspondingly fluorescent changes. Systematic optimization of parameters in "Signal-on" mode were carried out, the choice of QDNA length, the hybridization site of a small supplementary DNA (SDNA) stabilizer, and the existence of Mg2+cation played essential roles for the high sensitivity. With the optimized conditions, a convenient and sensitive determination of 1 nM rHuEPO-αwithout any preconcentration was achieved with the LOD of 0.4 nM.
In summary, we successfully developed the agglutinin-directed affinity chromatographic SELEX to obtain a series of ssDNA aptamers with high affinity and specificity for rHuEPO-α. The structure and function researches were carried out with aptamer 807 and 813, the chief binding sites were determined preliminaryly. The best aptamer 807-39nt with Kd value of 39±27 nM proposed here can be set as powerful biosensors to facilitate new methods for rHuEPO-αand develop effective diagnostic reagent for rHuEPO-αas an alternative approach to antibody.
基于rHuEPO具有提高红细胞运输氧的药理功能,在耐力比赛中,存在运动员滥用此类药物以提高比赛成绩的可能性,因此世界反兴奋剂组织(WADA)的违禁药物清单中已明确将其列为肽类激素兴奋剂。目前开展的针对rHuEPO的检测方法,主要基于抗体作为传感或富集元件,以免疫分析或质谱分析为手段实现rHuEPO的检测。但抗体自身具有批间差异性、免疫原性和热不稳定性的局限性,将会阻碍目前基于抗体的免疫分析方法的特异性和灵敏度的提高。因此,针对抗体外新识别元件的研制,将对rHuEPO的分析检测提供更加有效的新思路和新途径。
适配体是一类可折叠形成一定三维结构用于特异性识别靶分子的单链DNA,RNA或修饰后的核酸分子,与抗体有类似的识别特性,但较抗体相比特异性更强,并具有无免疫原性、易从体外化学途径制备、及热稳定性更强的优点,这就为开发rHuEPO-α的高亲和力、高特异性适配体提供了新思路。因此实验设计采用凝集素导向的指数富集配基的系统进化(SELEX)技术,从全长80个碱基的单链脱氧寡核苷酸文库中,经8轮反复富集,成功筛选获得特异性识别rHuEPO-α的寡核苷酸适配体。并对筛选得到的部分高亲和力、高特异性的适配体进行结构分析。最终,基于具有天然茎环式二级结构的适配体807-39 nt,建立了一种新型的适配体型分子信标检测策略,实现rHuEPO-α的高灵敏度高特异性的检测。
本论文共分为四章。第一章为文献综述,从促红细胞生成素的功能／用途及研究概况、SELEX筛选技术的发展、分子信标的原理及设计策略三方面进行概括,引用文献115篇。
第二章详细介绍了rHuEPO-α的特异性寡核苷酸适配体的筛选过程。实验中采用凝集素导向的亲和树脂SELEX技术,将rHuEPO-α的唾液酸糖基部分固定后,实现针对其氨基酸序列的筛选,经N-乙酰葡萄糖胺特异性洗脱寡核苷酸与蛋白的复合物,经过8轮筛选后各轮(收敛)寡核苷酸文库的富集效率由13.8%提高到64.0%,同时各轮文库对于rHuEPO-α的选择性也由1.4增加到10.6。随后,对于第8轮富集文库的单克隆序列进行了序列比对分析,主要可以分为四个家族。Mfold模拟所得序列的二级结构主要呈现三大特征：紧密的茎环结构、多分支的茎环结构、少或无茎环结构。其中部分单克隆适配体的解离常数在几十nM到几百nM之间,亲和力最高的为适配体807(Kd=82±32 nM)。
第三章为rHuEPO-α的特异性寡核苷酸适配体的截短序列研究。对于具有相对较低的吉布斯自由能和较低解离常数的适配体807和813,进行了进一步序列截短分析研究,以优化和寻求最短的有效结合序列长度。在第8轮文库中所占比例最高的序列807,其完全随机区807-39nt具有紧密的茎环结构,解离常数低于全长序列807,说明807-39nt的茎环结构对于复合物的形成有着重要作用。序列813的最短有效截短长度为813-42nt,其3’引物端的部分序列参与了随机区的茎部配对,并有利于复合物的形成。
适配体807-39nt具有发卡式二级结构,主要由一富含G碱基的环部和长度为5对碱基的茎部组成。对于其茎部／环部的系列长度改变／碱基突变等研究表明,其环部为与靶分子rHuEPO-α相互作用的主要结合位点,而茎部的主要作用为稳定复合物的形成。并进一步针对靶蛋白的结合位点进行研究,去糖基化实验结果表明,当糖蛋白rHuEPO-α去除N连接的末端糖基后,仍可以与适配体807-39nt发生特异性的相互作用。因此,可初步推测,对于适配体807-39nt,蛋白rHuEPO-α与其作用的主要结合部位为其氨基酸序列。适配体807-39nt可以识别商品化注射液(益比奥和罗可曼)中不同类型的rHuEPO,亦从另一方面说明了rHuEPO的氨基酸序列为适配体807-39nt的主要作用区域。同时膀胱正常上皮组织的免疫组化实验证实适配体807-39nt特异性优于rHuEPO-α的单克隆抗体AE7A5,并发现适配体807-39nt与膀胱癌上皮组织间具有特异性相互作用。
适配体807-39nt具有天然的茎环结构,在第四章中,基于此点我们建立了特异性识别rHuEPO-α的适配体型分子信标检测策略。分别构建"Signal-on"和"Signal-off"两种分子信标模式,依据适配体荧光信号的变化判断其结合靶分子rHuEPO-α前后分子构象的改变。对于"Signal-on"体系,淬灭DNA序列的长度、稳定DNA的杂交位置和金属离子Mg2+的浓度是其获得高灵敏度的关键因素,在优化后的条件下,未经任何富集手段可实现1 nM rHuEPO-α的直接快速灵敏检测,其LOD为0.4 nM。
综上所述,本论文成功建立了凝集素导向的亲和SELEX技术,用于筛选rHuEPO-α的高亲和力、高特异性的ssDNA适配体。对筛选得到的适配体807和813进行了结构功能研究,并初步判定了其和rHuEPO-α的主要作用位点。其中亲和力和特异性最佳的适配体807-39nt(Kd=39±27 nM)有望成为新的传感元件,可为拓宽现有的rHuEPO-α检测方法,开发有效的针对rHuEPO-α诊断试剂提供了除抗体之外的新途径。
Erythropoietin (EPO) is a kind of significant occurring hematopoietic growth factor and produced mainly in the kidney, which has the ability of promoting the proliferation and differentiation of erythroid progenitor cells and enriches the count of oxygen-carrying red blood cells. In recent years, the technique of transfection of EPO gene in mammalian cell lines makes large scale production of biologically active recombinant human EPO (rHuEPO) convenient with the development of genetic engineering. In all kinds of EPO preparations, rHuEPO-αand rHuEPO-β, have been extensively used in clinic to treat for anemia associated with renal disease and cancer.
It is possible for rHuEPO-αto be abused in endurance sports to improve the performance by many athletes for its pharmacological action of enhancing the count of oxygen-carrying red blood cells. As a kind of well-known doping peptide hormone, erythropoiesis-stimulating agents are labeled clearly in the prohibited list of World Anti-Doping Code. Immunological or mass spectrometric-based techniques are mainly employed for its analysis with the aid of antibodies as a sensing and enrichment element. However, the limitation of anti-EPO antibody, such as batch-to-batch variability, immunogenicity and thermolability, will hinder the improvement of specificity and sensitivity in immunoassays. The effectively analytical strategies are greatly needed for detection of rHuEPO with the development of new recognition element.
Aptamers are single-stranded DNA, RNA, or modified nucleic acids that have the ability to form defined tertiary structures for specific target binding. They have the similar recognition characterization as antibody and have no immunogenicity, easy preparation by chemical synthesis in vitro and strong thermal stability, these unique merits of aptamer are providing a promising alternative way to pursue affinity ligands with high affinity and high specificity for rHuEPO-α. So our group adopted agglutinin-directed Systematic Evolution of Ligands by EXponential enrichment (SELEX) technique to generate aptamers for rHuEPO-α, and a series of aptamers with specifity for rHuEPO-a were successfully obtained from 80nt ssDNA library after 8 selection rounds. Some aptamers with high affinity and specificity were chosen to carry out structure analysis. Finally, a new pattern of aptameric molecular beacon strategy was developed for the detection of rHuEPO-a basd on aptamer 807-39nt with naturally stem-loop secondary structure.
The dissertation contains four parts, the first part is an overview on three aspects including instruction of function and purpose of EPO, development of SELEX technology and principle of molecular beacon. Total 115 references were cited.
In the second part, the selection procedure of aptamer with high affinity and specificity for rHuEPO-a were introduced in detail. The wheat germ agglutinin (WGA)-directed affinity chromatographic SELEX was adopted to bind the sialic acids moiety of rHuEPO-a, and exposing peptide chains for selection, then a high-concentrated N-acetylglucosamine solution was applied to elute the complex of rHuEPO-a and ssDNAs by the displacement of sialic acids moiety from WGA-Sepharose. After 8 rounds of selection, the enrichment efficiency of oligonucleotides was obviously improved from 13.8% to 64.0% by determination of the percentage of bound ssDNAs and the selectivity of ssDNAs for rHuEPO-a was increased from 1.4 to 10.6. The clones sequenced from the 8th pool were divided into four families, the characteristics of these secondary structure present tight hairpins, multi-branched hairpins and few or no hairpins. The dissociation constants of some sequences were nM level, the sequence 807 can bind with rHuEPO-a with the highest affinity and specificity (Kd=82±32 nM).
The third part is mainly on the truncation research of aptamers for rHuEPO-a. The sequences 807 and 813 with relative lower Gibbs' energy and nanomole dissociation constant were selected to carry out truncation research in order to optimize and find the effective binding motif. The random sequence 807-39nt with tight hairpin structure truncated from the most frequently repeated sequence 807 exhibited by far the highest binding for rHuEPO-a, which can account for the superior binding capacity of the hairpin structure. The aptamer 813-42nt was the shortest motif truncated from 813 for binding with rHuEPO-a, EMS A results indicate that the stem forming between the region sequence and partly 3'primer region might facilitate the binding capacity.
The secondary structure of 807-39nt is composed of a G-rich loop and a 5bp stem. A series of stem and loop variation experiments show that the recognition is occurred mainly in the loop part and the stem is functioned as a support to fix an enclosed loop in order to stabilize the complex of rHuEPO-α. Also the binding site of target rHuEPO-αwas further determined by the deglycosylation experiment, rHuEPO-αwithout N-glycosylation still remain the binding ability with 807-39nt. We might speculate that the binding site of protein is mainly the amino acid sequence, which is further confirmed by the binding between 807-39nt and Injection of Yi Bi Ao or Recormn. Also the results of immunohistochemistry experiments on normal urothelium showed that the specificity of aptamer 807-39nt was superior than anti-rHuEPO-αmAb AE7A5, and the aptamer 807-39nt has the ability to bind with malignant urothelium.
In the fourth part, we designed the aptameric molecular beacon (MB) for rHuEPO-αdetection, based on the stem-loop secondary structure of 807-39nt. Both "Signal-on" and "Signal-off' MB modes were developed, respectively, in which the conformational alteration of aptamer before and after binding to rHuEPO-a can be demonstrated in terms of the correspondingly fluorescent changes. Systematic optimization of parameters in "Signal-on" mode were carried out, the choice of QDNA length, the hybridization site of a small supplementary DNA (SDNA) stabilizer, and the existence of Mg2+cation played essential roles for the high sensitivity. With the optimized conditions, a convenient and sensitive determination of 1 nM rHuEPO-αwithout any preconcentration was achieved with the LOD of 0.4 nM.
In summary, we successfully developed the agglutinin-directed affinity chromatographic SELEX to obtain a series of ssDNA aptamers with high affinity and specificity for rHuEPO-α. The structure and function researches were carried out with aptamer 807 and 813, the chief binding sites were determined preliminaryly. The best aptamer 807-39nt with Kd value of 39±27 nM proposed here can be set as powerful biosensors to facilitate new methods for rHuEPO-αand develop effective diagnostic reagent for rHuEPO-αas an alternative approach to antibody.
引文
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