适配体的Post-SELEX筛选评价、表征及传感新方法研究
发布时间：2018-09-07 17:20
【摘要】：寡核苷酸适配体(Oligonucleotide aptamer,简称适配体,aptamer),系指经指数富集配基系统进化(Systematic Evolution of Ligands by EXponential enrichment,SELEX)技术筛选得到的一段短的单链寡核苷酸序列(ssDNA或RNA)。适配体通过折叠成一定的三维结构,经空间构型互补与靶分子形成高亲和力,高特异性结合,故又称为“化学抗体”。其与靶分子之间的分子识别作用与抗体极为相似,但特异性和亲和力甚至更高。与抗体相比,适配体稳定性好、能够进行大量的化学合成、并且无免疫原性、无毒性。另外,适配体具有核酸自身变性复性快速可逆、易功能化修饰与标记、及作为优良的纳米器件等诸多特点,是一类重要的“明星分子”。随着新型SELEX筛选技术、高通量测序、生物芯片等序列活性评价技术的快速应用,所筛选出来的适配体分子种类逐年增多,作用的靶分子范围从离子、小分子、转录因子、蛋白(酶),到细胞、组织、细菌、病毒等等,也更加广泛。适配体在分析传感、临床诊断和治疗方面存在巨大潜力。然而,到目前为止,仅有少量适配体进入到临床试验研究阶段,仅有一种适配体药物经FDA批准上市,即治疗老年相关性黄斑病变的哌加他尼。做为商业化临床诊断元件的适配体亦寥寥无几,此点与蓬勃发展的适配体研究趋势并不匹配。其主要技术瓶颈应在于其自身二三级结构不清、与药靶的相互作用机制不明、体内生物利用度尚待提高等方面。从分子相互作用基础层面而言,大多数适配体存在结构上的易变性、多样性,在与靶分子发生特异性的亲和识别时,多以“自适应”形成优势构型,但大多数适配体—靶分子的精确结构模型尚未得到,多数识别模式的判别具有片面性、经验性。另外,经SELEX技术筛选得到的适配体全长序列中,仅有部分序列存在亲和活性,亟需发展有效的技术方法指导结构剪裁及修饰,从中寻找亲和性更高、特异性更强、序列更短的适配体,除去冗余序列,提高适配体识别研究的容错度、通用性和稳定性。这就使得SELEX后优化(Post-SELEX)成为研究“热点”。Post-SELEX中多采用试错法,依据核酸二级结构及自由能(ΔG)的经验性判断,对适配体序列进行较为随机的剪裁或定点突变。其主要缺点在于随机方法效率较低、难度较大,尚无系统的理论指导和较好的解决方案。本学位论文的主要内容则针对上述post-SELEX中的基础科学问题进行了深入研究,共分为五章。第一章为前言(文献综述)部分,概述了适配体的特点、应用前景以及适配体研究存在的主要问题;对适配体后筛选优化途径、以及相应的相互作用评价优化方法进行了总结和评述;以及对适配体结构表征方法、适配体做为分析传感元件的应用现状进行了综述。最后提出本论文的立题依据及主要的研究内容。第二章为一种步进型组合文库的构建及基于SPR的亲和评价优化方法研究。在本章中,我们基于组合化学思想,以重组人促红细胞生成素α(EPO-α)的适配体807-39nt为模式分子,发明设计了四组步进型序列,构成新型的多方向、步进型适配体小型组合文库。该文库包含向左收缩、向右收缩、两端收缩和两端扩展等四组分子群,共35条序列。并开发了免标记SPR方法,对上述适配体序列进行快速、高效的筛选评价。最终得到了最短适配体—In27,仅保留了初始适配体39nt二级结构中的环部部分,但亲和活性与之相当。从而证实了该小型步进型组合文库构建的合理性和高效性。第三章为最短适配体In27的简并序列及结合位点研究。本章使用定点碱基突变的方法,结合结构域划分,筛选评价得到In27序列中对结合产生较大影响的关键碱基。考虑到In27仍然保留了适配体39nt中富含连续鸟嘌呤核苷酸的序列,符合G四聚体(GQ)形成的一般原则,我们在圆二色谱(CD)表征的基础上,将In27上的碱基按-GG(G)-骨架为间隔,划分为D1~D5个结构域,并构建了各定点突变分子群,共6组,分别命名为D1、D2、D34、D5、GQ和简并序列定点突变分子群,共32条序列。我们使用表面等离子共振(SPR)方法筛选评价,从该定点突变分子群中最终得到In27的简并序列,从而明晰了最短适配体In27的关键作用碱基。进一步,我们采用亲和竞争作用分析,界定了多种亲和识别分子,如适配体39nt及In27、EPO受体(EPOR)、EPO的单克隆抗体以及麦胚凝集素(WGA),在EPO-α上的结合作用模式。上述研究即为适配体的结构作用特点研究及适配体做为分析传感元件的成功应用奠定了基础。第四章为多种光谱技术对于适配体In27的活性高级结构特征研究。适配体的结构具有多样性,其“自适应”式优势构型较易受到包括pH、阳离子类型、稳定保护剂、甚至界面等在内的微环境的影响与调控,这也是大多数适配体和靶分子间相互作用特点尚待厘清和阐明的主要原因之一。本章发展和使用SPR、CD及荧光探针等多种光谱技术,研究和明确了In27的活性与构型间的关系。我们发现阳离子Na+的存在和浓度是影响In27产生特异性亲和识别的关键因素;Na+诱导下In27形成了平行/反平行的混合GQ构型;两种GQ荧光探针硫磺素T(ThT)和N-甲基间卟啉IX(NMM)在该体系中可用做一对特征性的荧光探针,均可用于表征In27的活性高级构型。EPO-α的加入可与ThT和NMM形成竞争,从而便于利用这两种GQ荧光探针创建活性EPO-α的“光开关”型检测方法。上述研究为阐明新型适配体的活性构型提供了有效的技术组合手段,亦为分析、传感、诊断等应用领域提供了一类作用特点清晰的适配体元件。第五章为基于适配体的免标记检测方法研究。生物膜干涉技术(BLI)是一种新型的表面传感技术,具有免标记、灵敏、快速、作用模式多样等特点。本章中,我们将生物素化的适配体39nt或In27固定于链霉亲和素(SA)传感芯片上,优化了用于表面固定的适配体元件的取向、浓度、非特异性作用的克服、再生条件等因素,并利用WGA和适配体作用于EPO-α的不同位点的特征,构建信号放大传感体系,用于溶液中EPO-α蛋白的灵敏检测。同时比较了SPR和BLI技术方法的异同并阐述可能原因。本研究拓宽了适配体做为分析传感元件的应用范围。总之,本学位论文立意于创建创新性的小型多方向步进型适配体组合文库,从而高效地筛选优化得到了最短适配体—In27,并在明确其关键碱基位点的贡献基础上构建简并序列。以多种光谱学技术手段,明确和简化得到影响适配体活性构型的关键溶液微环境因素,深入阐明了Na+诱导下的适配体In27的活性高级结构形成特点。最后,基于适配体实现了EPO-α蛋白的免标记、高灵敏检测。在为分析、传感、诊断等应用领域提供了一类作用特点清晰的适配体元件的同时,也为优化适配体结构,阐述适配体与靶蛋白相互作用时结构特征等提供了有效的技术手段。
[Abstract]:Oligonucleotide aptamer (aptamer) refers to a short single-stranded oligonucleotide sequence (ssDNA or RNA) screened by the technique of Systematic Evolution of Ligands by EXponential enrichment (SELEX). Interconfiguration complementarity is also called "chemical antibody" because of its high affinity and specific binding with target molecules. Its molecular recognition with target molecules is very similar to antibodies, but its specificity and affinity are even higher. In addition, aptamers are a class of important "star molecules" because of their fast and reversible denaturation and renaturation of nucleic acids, easy functionalization, modification and labeling, and as excellent nanodevices. With the rapid application of new SELEX screening techniques, high throughput sequencing, biochip and other sequencing activity evaluation techniques, the aptamers have been screened out. As the number of somatic molecules increases year by year, the range of target molecules is wider, ranging from ions, small molecules, transcription factors, proteins (enzymes), to cells, tissues, bacteria, viruses and so on. Only one of the aptamer drugs approved by the FDA, pigaetanil, for the treatment of age-related macular disease, has been commercially available as an aptamer for clinical diagnosis, which does not match the booming trend in aptamer research. The main technical bottleneck should be its unclear secondary and tertiary structure and its interaction with drug targets. From the basic level of molecular interaction, most aptamers have structural variability and diversity. When specific affinity recognition occurs with the target molecule, the dominant configuration is formed by "self-adaptation", but most aptamers-target molecule accurate structure model. In addition, only some of the full-length sequences of aptamers screened by SELEX technology have affinity activity, so it is urgent to develop effective technical methods to guide the structure tailoring and modification, and to find the aptamers with higher affinity, specificity and shorter sequences. Redundant sequences are removed to improve the fault tolerance, universality and stability of aptamer recognition, which makes post-SELEX become a hot spot of research. The main drawbacks of this dissertation are that random methods are inefficient and difficult, and there is no systematic theoretical guidance and a better solution. The main contents of this dissertation focus on the basic scientific problems in post-SELEX, which are divided into five chapters. The prospects and the main problems of aptamer research are summarized and reviewed. The methods of aptamer post-screening optimization and the corresponding interaction evaluation optimization methods are summarized and reviewed. The methods of aptamer structure characterization and the application status of aptamer as analytical sensor are summarized. Finally, the basis and main points of this paper are put forward. The second chapter is about the construction of a step-by-step combinatorial library and the optimization of affinity evaluation based on SPR. In this chapter, based on the idea of combinatorial chemistry, we invented and designed four groups of step-by-step sequences to form a new multi-direction, step-by-step sequence with the aptamer 807-39nt of recombinant human erythropoietin-alpha (EPO-alpha) as the model molecule. The library contains 35 sequences of four groups, namely, left-contraction, right-contraction, two-end contraction and two-end extension. A label-free SPR method was developed to screen and evaluate the aptamer sequences quickly and efficiently. Finally, the shortest aptamer, In27, was obtained, with only 39nt2 retained as the initial aptamer. In the third chapter, the degenerate sequence and binding sites of the shortest aptamer In27 were studied. In this chapter, site-directed base mutation and domain partition were used to screen and evaluate the pairing of In27 sequences. Considering that In27 still retains the sequence rich in continuous guanine nucleotides in aptamer 39nt and conforms to the general principle of G-tetramer (GQ) formation, we divided the bases on In27 into five domains D1-D5 by - GG (G) - skeleton separation on the basis of circular dichroism (CD) characterization. Six groups of site-directed mutants, named D1, D2, D34, D5, GQ and degenerate sequence site-directed mutants, were selected and evaluated by surface plasmon resonance (SPR). The degenerate sequence of In27 was finally obtained from the site-directed mutant population, thus clarifying the key functional base of the shortest aptamer In27. Many affinity recognition molecules, such as aptamers 39nt and In27, EPO receptors (EPOR), monoclonal antibodies against EPO, and wheat germ agglutinin (WGA), have been identified by affinity-competition analysis. These studies will lay a foundation for the study of the structural characteristics of aptamers and the successful application of aptamers as analytical sensors. Chapter 4 is the study of the active high-level structural characteristics of aptamer In27 by various spectroscopic techniques. The structure of aptamers is diverse, and their "adaptive" dominant configurations are more susceptible to the influence and regulation of microenvironment including pH, cationic type, stabilizer and protector, and even interface, which are also the majority of aptamers and interfaces. In this chapter, we have developed and used SPR, CD and fluorescent probes to study and clarify the relationship between the activity and configuration of In27. We found that the presence and concentration of cationic Na + are the key factors affecting the specific affinity recognition of In27. Two kinds of GQ fluorescent probes, sulfur T (ThT) and N-methylmesoporphyrin IX (NMM), can be used as a pair of characteristic fluorescent probes in the system, and both of them can be used to characterize the active high-order configuration of In27. The addition of EPO-a can compete with ThT and NMM, thus facilitating the use of these two GQ fluorescent probes. The above studies provide an effective combination of techniques for elucidating the active configuration of novel aptamers and a class of aptamer elements with distinct functional characteristics for analysis, sensing, diagnosis and other applications. Chapter 5 is the study of aptamer-based label-free detection methods. Interferometry (BLI) is a new kind of surface sensing technology, which is label-free, sensitive, fast and has a variety of action modes. In this chapter, we immobilize the biotinylated aptamers 39nt or In27 on the streptavidin (SA) sensor chip to optimize the orientation, concentration, and nonspecific effect of aptamers for surface immobilization. Based on the characteristics of different sites of EPO-alpha acted by WGA and aptamers, a signal amplification sensing system was constructed for sensitive detection of EPO-alpha protein in solution. The similarities and differences between SPR and BLI techniques were compared and the possible reasons were explained. Therefore, this dissertation aims to create a small, innovative, multi-directional, step-by-step aptamer library to efficiently screen and optimize the shortest aptamer, In27, and to construct degenerate sequences based on the contribution of its key base sites. The key solution micro-environment factors of the model A further elucidate the formation characteristics of the active high-level structure of aptamer In27 induced by Na +. Finally, the label-free and highly sensitive detection of EPO-alpha protein is realized based on aptamers. The structure of aptamers and their interaction with target proteins provide effective technical means.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78
，
本文编号：2228913
[Abstract]:Oligonucleotide aptamer (aptamer) refers to a short single-stranded oligonucleotide sequence (ssDNA or RNA) screened by the technique of Systematic Evolution of Ligands by EXponential enrichment (SELEX). Interconfiguration complementarity is also called "chemical antibody" because of its high affinity and specific binding with target molecules. Its molecular recognition with target molecules is very similar to antibodies, but its specificity and affinity are even higher. In addition, aptamers are a class of important "star molecules" because of their fast and reversible denaturation and renaturation of nucleic acids, easy functionalization, modification and labeling, and as excellent nanodevices. With the rapid application of new SELEX screening techniques, high throughput sequencing, biochip and other sequencing activity evaluation techniques, the aptamers have been screened out. As the number of somatic molecules increases year by year, the range of target molecules is wider, ranging from ions, small molecules, transcription factors, proteins (enzymes), to cells, tissues, bacteria, viruses and so on. Only one of the aptamer drugs approved by the FDA, pigaetanil, for the treatment of age-related macular disease, has been commercially available as an aptamer for clinical diagnosis, which does not match the booming trend in aptamer research. The main technical bottleneck should be its unclear secondary and tertiary structure and its interaction with drug targets. From the basic level of molecular interaction, most aptamers have structural variability and diversity. When specific affinity recognition occurs with the target molecule, the dominant configuration is formed by "self-adaptation", but most aptamers-target molecule accurate structure model. In addition, only some of the full-length sequences of aptamers screened by SELEX technology have affinity activity, so it is urgent to develop effective technical methods to guide the structure tailoring and modification, and to find the aptamers with higher affinity, specificity and shorter sequences. Redundant sequences are removed to improve the fault tolerance, universality and stability of aptamer recognition, which makes post-SELEX become a hot spot of research. The main drawbacks of this dissertation are that random methods are inefficient and difficult, and there is no systematic theoretical guidance and a better solution. The main contents of this dissertation focus on the basic scientific problems in post-SELEX, which are divided into five chapters. The prospects and the main problems of aptamer research are summarized and reviewed. The methods of aptamer post-screening optimization and the corresponding interaction evaluation optimization methods are summarized and reviewed. The methods of aptamer structure characterization and the application status of aptamer as analytical sensor are summarized. Finally, the basis and main points of this paper are put forward. The second chapter is about the construction of a step-by-step combinatorial library and the optimization of affinity evaluation based on SPR. In this chapter, based on the idea of combinatorial chemistry, we invented and designed four groups of step-by-step sequences to form a new multi-direction, step-by-step sequence with the aptamer 807-39nt of recombinant human erythropoietin-alpha (EPO-alpha) as the model molecule. The library contains 35 sequences of four groups, namely, left-contraction, right-contraction, two-end contraction and two-end extension. A label-free SPR method was developed to screen and evaluate the aptamer sequences quickly and efficiently. Finally, the shortest aptamer, In27, was obtained, with only 39nt2 retained as the initial aptamer. In the third chapter, the degenerate sequence and binding sites of the shortest aptamer In27 were studied. In this chapter, site-directed base mutation and domain partition were used to screen and evaluate the pairing of In27 sequences. Considering that In27 still retains the sequence rich in continuous guanine nucleotides in aptamer 39nt and conforms to the general principle of G-tetramer (GQ) formation, we divided the bases on In27 into five domains D1-D5 by - GG (G) - skeleton separation on the basis of circular dichroism (CD) characterization. Six groups of site-directed mutants, named D1, D2, D34, D5, GQ and degenerate sequence site-directed mutants, were selected and evaluated by surface plasmon resonance (SPR). The degenerate sequence of In27 was finally obtained from the site-directed mutant population, thus clarifying the key functional base of the shortest aptamer In27. Many affinity recognition molecules, such as aptamers 39nt and In27, EPO receptors (EPOR), monoclonal antibodies against EPO, and wheat germ agglutinin (WGA), have been identified by affinity-competition analysis. These studies will lay a foundation for the study of the structural characteristics of aptamers and the successful application of aptamers as analytical sensors. Chapter 4 is the study of the active high-level structural characteristics of aptamer In27 by various spectroscopic techniques. The structure of aptamers is diverse, and their "adaptive" dominant configurations are more susceptible to the influence and regulation of microenvironment including pH, cationic type, stabilizer and protector, and even interface, which are also the majority of aptamers and interfaces. In this chapter, we have developed and used SPR, CD and fluorescent probes to study and clarify the relationship between the activity and configuration of In27. We found that the presence and concentration of cationic Na + are the key factors affecting the specific affinity recognition of In27. Two kinds of GQ fluorescent probes, sulfur T (ThT) and N-methylmesoporphyrin IX (NMM), can be used as a pair of characteristic fluorescent probes in the system, and both of them can be used to characterize the active high-order configuration of In27. The addition of EPO-a can compete with ThT and NMM, thus facilitating the use of these two GQ fluorescent probes. The above studies provide an effective combination of techniques for elucidating the active configuration of novel aptamers and a class of aptamer elements with distinct functional characteristics for analysis, sensing, diagnosis and other applications. Chapter 5 is the study of aptamer-based label-free detection methods. Interferometry (BLI) is a new kind of surface sensing technology, which is label-free, sensitive, fast and has a variety of action modes. In this chapter, we immobilize the biotinylated aptamers 39nt or In27 on the streptavidin (SA) sensor chip to optimize the orientation, concentration, and nonspecific effect of aptamers for surface immobilization. Based on the characteristics of different sites of EPO-alpha acted by WGA and aptamers, a signal amplification sensing system was constructed for sensitive detection of EPO-alpha protein in solution. The similarities and differences between SPR and BLI techniques were compared and the possible reasons were explained. Therefore, this dissertation aims to create a small, innovative, multi-directional, step-by-step aptamer library to efficiently screen and optimize the shortest aptamer, In27, and to construct degenerate sequences based on the contribution of its key base sites. The key solution micro-environment factors of the model A further elucidate the formation characteristics of the active high-level structure of aptamer In27 induced by Na +. Finally, the label-free and highly sensitive detection of EPO-alpha protein is realized based on aptamers. The structure of aptamers and their interaction with target proteins provide effective technical means.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78
，
本文编号：2228913
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