適配體的Post-SELEX篩選評價、表征及傳感新方法研究
發(fā)布時間：2018-09-07 17:20
【摘要】：寡核苷酸適配體(Oligonucleotide aptamer,簡稱適配體,aptamer),系指經(jīng)指數(shù)富集配基系統(tǒng)進(jìn)化(Systematic Evolution of Ligands by EXponential enrichment,SELEX)技術(shù)篩選得到的一段短的單鏈寡核苷酸序列(ssDNA或RNA)。適配體通過折疊成一定的三維結(jié)構(gòu),經(jīng)空間構(gòu)型互補(bǔ)與靶分子形成高親和力,高特異性結(jié)合,故又稱為“化學(xué)抗體”。其與靶分子之間的分子識別作用與抗體極為相似,但特異性和親和力甚至更高。與抗體相比,適配體穩(wěn)定性好、能夠進(jìn)行大量的化學(xué)合成、并且無免疫原性、無毒性。另外,適配體具有核酸自身變性復(fù)性快速可逆、易功能化修飾與標(biāo)記、及作為優(yōu)良的納米器件等諸多特點(diǎn),是一類重要的“明星分子”。隨著新型SELEX篩選技術(shù)、高通量測序、生物芯片等序列活性評價技術(shù)的快速應(yīng)用,所篩選出來的適配體分子種類逐年增多,作用的靶分子范圍從離子、小分子、轉(zhuǎn)錄因子、蛋白(酶),到細(xì)胞、組織、細(xì)菌、病毒等等,也更加廣泛。適配體在分析傳感、臨床診斷和治療方面存在巨大潛力。然而,到目前為止,僅有少量適配體進(jìn)入到臨床試驗(yàn)研究階段,僅有一種適配體藥物經(jīng)FDA批準(zhǔn)上市,即治療老年相關(guān)性黃斑病變的哌加他尼。做為商業(yè)化臨床診斷元件的適配體亦寥寥無幾,此點(diǎn)與蓬勃發(fā)展的適配體研究趨勢并不匹配。其主要技術(shù)瓶頸應(yīng)在于其自身二三級結(jié)構(gòu)不清、與藥靶的相互作用機(jī)制不明、體內(nèi)生物利用度尚待提高等方面。從分子相互作用基礎(chǔ)層面而言,大多數(shù)適配體存在結(jié)構(gòu)上的易變性、多樣性,在與靶分子發(fā)生特異性的親和識別時,多以“自適應(yīng)”形成優(yōu)勢構(gòu)型,但大多數(shù)適配體—靶分子的精確結(jié)構(gòu)模型尚未得到,多數(shù)識別模式的判別具有片面性、經(jīng)驗(yàn)性。另外,經(jīng)SELEX技術(shù)篩選得到的適配體全長序列中,僅有部分序列存在親和活性,亟需發(fā)展有效的技術(shù)方法指導(dǎo)結(jié)構(gòu)剪裁及修飾,從中尋找親和性更高、特異性更強(qiáng)、序列更短的適配體,除去冗余序列,提高適配體識別研究的容錯度、通用性和穩(wěn)定性。這就使得SELEX后優(yōu)化(Post-SELEX)成為研究“熱點(diǎn)”。Post-SELEX中多采用試錯法,依據(jù)核酸二級結(jié)構(gòu)及自由能(ΔG)的經(jīng)驗(yàn)性判斷,對適配體序列進(jìn)行較為隨機(jī)的剪裁或定點(diǎn)突變。其主要缺點(diǎn)在于隨機(jī)方法效率較低、難度較大,尚無系統(tǒng)的理論指導(dǎo)和較好的解決方案。本學(xué)位論文的主要內(nèi)容則針對上述post-SELEX中的基礎(chǔ)科學(xué)問題進(jìn)行了深入研究,共分為五章。第一章為前言(文獻(xiàn)綜述)部分,概述了適配體的特點(diǎn)、應(yīng)用前景以及適配體研究存在的主要問題;對適配體后篩選優(yōu)化途徑、以及相應(yīng)的相互作用評價優(yōu)化方法進(jìn)行了總結(jié)和評述;以及對適配體結(jié)構(gòu)表征方法、適配體做為分析傳感元件的應(yīng)用現(xiàn)狀進(jìn)行了綜述。最后提出本論文的立題依據(jù)及主要的研究內(nèi)容。第二章為一種步進(jìn)型組合文庫的構(gòu)建及基于SPR的親和評價優(yōu)化方法研究。在本章中,我們基于組合化學(xué)思想,以重組人促紅細(xì)胞生成素α(EPO-α)的適配體807-39nt為模式分子,發(fā)明設(shè)計了四組步進(jìn)型序列,構(gòu)成新型的多方向、步進(jìn)型適配體小型組合文庫。該文庫包含向左收縮、向右收縮、兩端收縮和兩端擴(kuò)展等四組分子群,共35條序列。并開發(fā)了免標(biāo)記SPR方法,對上述適配體序列進(jìn)行快速、高效的篩選評價。最終得到了最短適配體—In27,僅保留了初始適配體39nt二級結(jié)構(gòu)中的環(huán)部部分,但親和活性與之相當(dāng)。從而證實(shí)了該小型步進(jìn)型組合文庫構(gòu)建的合理性和高效性。第三章為最短適配體In27的簡并序列及結(jié)合位點(diǎn)研究。本章使用定點(diǎn)堿基突變的方法,結(jié)合結(jié)構(gòu)域劃分,篩選評價得到In27序列中對結(jié)合產(chǎn)生較大影響的關(guān)鍵堿基。考慮到In27仍然保留了適配體39nt中富含連續(xù)鳥嘌呤核苷酸的序列,符合G四聚體(GQ)形成的一般原則,我們在圓二色譜(CD)表征的基礎(chǔ)上,將In27上的堿基按-GG(G)-骨架為間隔,劃分為D1~D5個結(jié)構(gòu)域,并構(gòu)建了各定點(diǎn)突變分子群,共6組,分別命名為D1、D2、D34、D5、GQ和簡并序列定點(diǎn)突變分子群,共32條序列。我們使用表面等離子共振(SPR)方法篩選評價,從該定點(diǎn)突變分子群中最終得到In27的簡并序列,從而明晰了最短適配體In27的關(guān)鍵作用堿基。進(jìn)一步,我們采用親和競爭作用分析,界定了多種親和識別分子,如適配體39nt及In27、EPO受體(EPOR)、EPO的單克隆抗體以及麥胚凝集素(WGA),在EPO-α上的結(jié)合作用模式。上述研究即為適配體的結(jié)構(gòu)作用特點(diǎn)研究及適配體做為分析傳感元件的成功應(yīng)用奠定了基礎(chǔ)。第四章為多種光譜技術(shù)對于適配體In27的活性高級結(jié)構(gòu)特征研究。適配體的結(jié)構(gòu)具有多樣性,其“自適應(yīng)”式優(yōu)勢構(gòu)型較易受到包括pH、陽離子類型、穩(wěn)定保護(hù)劑、甚至界面等在內(nèi)的微環(huán)境的影響與調(diào)控,這也是大多數(shù)適配體和靶分子間相互作用特點(diǎn)尚待厘清和闡明的主要原因之一。本章發(fā)展和使用SPR、CD及熒光探針等多種光譜技術(shù),研究和明確了In27的活性與構(gòu)型間的關(guān)系。我們發(fā)現(xiàn)陽離子Na+的存在和濃度是影響In27產(chǎn)生特異性親和識別的關(guān)鍵因素;Na+誘導(dǎo)下In27形成了平行/反平行的混合GQ構(gòu)型;兩種GQ熒光探針硫磺素T(ThT)和N-甲基間卟啉IX(NMM)在該體系中可用做一對特征性的熒光探針,均可用于表征In27的活性高級構(gòu)型。EPO-α的加入可與ThT和NMM形成競爭,從而便于利用這兩種GQ熒光探針創(chuàng)建活性EPO-α的“光開關(guān)”型檢測方法。上述研究為闡明新型適配體的活性構(gòu)型提供了有效的技術(shù)組合手段,亦為分析、傳感、診斷等應(yīng)用領(lǐng)域提供了一類作用特點(diǎn)清晰的適配體元件。第五章為基于適配體的免標(biāo)記檢測方法研究。生物膜干涉技術(shù)(BLI)是一種新型的表面?zhèn)鞲屑夹g(shù),具有免標(biāo)記、靈敏、快速、作用模式多樣等特點(diǎn)。本章中,我們將生物素化的適配體39nt或In27固定于鏈霉親和素(SA)傳感芯片上,優(yōu)化了用于表面固定的適配體元件的取向、濃度、非特異性作用的克服、再生條件等因素,并利用WGA和適配體作用于EPO-α的不同位點(diǎn)的特征,構(gòu)建信號放大傳感體系,用于溶液中EPO-α蛋白的靈敏檢測。同時比較了SPR和BLI技術(shù)方法的異同并闡述可能原因。本研究拓寬了適配體做為分析傳感元件的應(yīng)用范圍�？傊�,本學(xué)位論文立意于創(chuàng)建創(chuàng)新性的小型多方向步進(jìn)型適配體組合文庫,從而高效地篩選優(yōu)化得到了最短適配體—In27,并在明確其關(guān)鍵堿基位點(diǎn)的貢獻(xiàn)基礎(chǔ)上構(gòu)建簡并序列。以多種光譜學(xué)技術(shù)手段,明確和簡化得到影響適配體活性構(gòu)型的關(guān)鍵溶液微環(huán)境因素,深入闡明了Na+誘導(dǎo)下的適配體In27的活性高級結(jié)構(gòu)形成特點(diǎn)。最后,基于適配體實(shí)現(xiàn)了EPO-α蛋白的免標(biāo)記、高靈敏檢測。在為分析、傳感、診斷等應(yīng)用領(lǐng)域提供了一類作用特點(diǎn)清晰的適配體元件的同時,也為優(yōu)化適配體結(jié)構(gòu),闡述適配體與靶蛋白相互作用時結(jié)構(gòu)特征等提供了有效的技術(shù)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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q78
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本文編號：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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q78
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本文編號：2228913
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