核酸适配体筛选的改良与优化策略

核酸适配体以其类似抗体的功能及诸多优于抗体的特点引起人们的广泛关注,如何利用指数富集的配基系统进化技术(SELEX技术)来高效地筛选核酸适配体是其应用的关键。结合近些年核酸适配体的研究进展,为提高筛选效率及核酸适配体性能,针对适配体筛选三大不同环节所做的尝试及改良进行相关的阐述,包括初始随机寡核苷酸文库的设计与修饰、筛选过程所用方法及次级文库的制备、筛选过程后(Post-SELEX)核酸适配体性能的优化等;特别比较了筛选方法包括毛细管电泳SELEX、细胞SELEX、磁珠SELEX、加尾SELEX、微流控SELEX、微阵列SELEX和高通量SELEX,以及次级文库的制备方法诸如不对称PCR法、λ核酸外切酶法、磁珠法、不等长PCR法、综合性方法等;对核酸适配体发展面临的问题及发展前景进行探讨,以期为相关科研人员的研究提供参考。     

综述与专论: 核酸适配体在分子医学中的应用

分子医学着眼于从疾病的分子层面出发,为个性化精准诊疗提供解决方案。然而,在众多疾病的诊疗中由于缺乏有力的分子识别工具,目前从分子水平上理解和研究疾病仍受到制约。核酸适配体是通过指数富集的配体系统进化（SELEX）技术在体外筛选得到的单链寡核苷酸,具有高选择性、高亲和力、易细胞内化、良好的组织渗透和快速的组织积累能力。近年来,由于其易合成、成本低、稳定性高且免疫原性低,核酸适配体作为分子工具应用于疾病的诊疗一体化受到广泛关注。本综述围绕分子医学中的核酸适配体,讨论了核酸适配体在疾病诊断中的应用,包括基于核酸适配体的肿瘤标志物发现、液体活检、分子成像。介绍了核酸适配体在癌症治疗中的应用包括基于核酸适配体的抑制剂、核酸适配体药物偶联物、纳米药物和核酸适配体介导的免疫治疗。最后对核酸适配体在临床诊疗和产业化面临的问题进行了讨论,包括基于应用场景的筛选方法、核酸适配体与靶标复合物结构、亲和力的机制以及核酸适配体在血液循环中的稳定性等方面。     

SELEX技术及近年研究进展

指数富集配体系统进化技术(Systematic evolution of ligand by exponential enrichment,SELEX)是近20年来出现的一种新型体外筛选技术。该技术将文库技术与筛选技术相结合,从核酸文库中筛选出的一段核酸分子:配基-适配子(aptamer);适配子与抗体类似,可以与相应靶物质特异性结合。SELEX技术筛选适配子的技术有快速的进步,而适配子也被应用到医学生物学等各个方面。在HIV等病毒学研究与治疗方面,SELEX的应用也有了可观的进展,多种病毒的相关适配子被筛选出来,为病毒相关疾病的防治提供了新的方向。本文主要就近三年来的SELEX技术发展状况及在应用上的发展进行简要综述。     

综述与专论: 核酸适配体筛选与亲和力评价技术主要研究方向、进展与挑战

核酸适配体是一类具有特异性分子识别能力的单链DNA或者RNA分子,通过指数富集的配体系统进化技术（SELEX）筛选得到。核酸适配体相比抗体具有热稳定性高、便于化学合成与修饰、免疫原性低等优点,在生物分析、生物医学、生物技术等众多领域引起广泛关注。高质量的核酸适配体是应用的基础,然而目前能够满足实际应用的核酸适配体数量还非常有限。如何获得高亲和力、高特异性、高体内稳定性的核酸适配体是核酸适配体领域的技术瓶颈。本文首先简单介绍了SELEX技术的基本原理和核酸库的设计、筛选过程监控、次级文库制备、测序和候选适配体筛选等关键步骤。接着归纳总结了30多年来核酸适配体筛选技术的6个主要研究方向、研究进展和局限性。这6个主要研究方向分别是提高适配体特异性的筛选方法、提高适配体稳定性（抗核酸酶降解能力）的筛选方法、快速筛选方法、复杂靶标适配体筛选方法、小分子靶标适配体筛选方法、提高适配体亲和力的筛选方法。其中快速筛选技术是长期以来持续关注的研究方向,几乎所有物理分离手段都已用于提高SELEX的筛选效率。最近,高效化学反应与SELEX技术的结合为核酸适配体的快速筛选提供了新的策略。本文随后对适合小分子靶标核酸适配体筛选的3类方法进展和存在的问题进行了重点评述。这3类方法分别是基于靶标固定的筛选技术、基于文库固定的筛选技术（捕获-SELEX,Capture-SELEX）和均相筛选技术（氧化石墨烯-SELEX,GO-SELEX）。基于靶标固定的筛选技术尽管存在空间位阻等众多问题,由于其操作的简单性,目前依然应用广泛。近年来Capture-SELEX应用广泛。结合36种靶标适配体的筛选实验条件（文库设计、正筛靶标浓度、负筛靶标的选择和浓度）和所获得的适配体的亲和力（K_D,解离常数,dissociation constant）和特异性,对Capture-SELEX的实验条件与适配体性能的关系进行了讨论。统计数据表明,降低正筛靶标浓度有利于提高适配体的亲和力,但不是必要条件。负筛选是目前提高适配体特异性的主要技术手段,但适配体的特异性还不能满足实际需求。负筛选靶标及其浓度的选择差异很大,而且36种靶标中有20种靶标的适配体筛选没有进行负筛选。如何提高核酸适配体的特异性是目前小分子靶标核酸适配体所面临的难题,急需寻找新的策略。本文还列表归纳了近三年利用GO-SELEX进行的13种小分子靶标的实验条件和所获得的适配体的K_D和特异性。统计数据表明,GO-SELEX比Capture-SELEX所需要的筛选轮数少,两种方法所获得的适配体的亲和力多在纳摩尔每升水平。Capture-SELEX相对较低的筛选效率应该主要由于文库的自解离问题。核酸适配体的亲和力评价是候选核酸适配体结构与性能评价的重要组成部分。常用的核酸适配体亲和力评价技术包括基于分离、基于固定、均相体系三大类十多种方法。假阳性和假阴性是各种评价技术都有可能存在的问题。本文以纳米金比色法和等温热滴定技术为例评述技术进展,讨论导致不同亲和力评价技术结果不一致性问题的根本原因。本文最后对核酸适配体筛选技术、亲和力评价技术和技术的标准化的未来发展趋势进行了展望。     

细胞特异性核酸适配体筛选新策略的研究进展

核酸适配体(aptamer)是在体外采用指数富集的配基系统进化技术(systematic evolution of ligands by exponential enrichment, SELEX)从人工合成的随机寡核苷酸文库中筛选得到的一段寡核苷酸序列(RNA或DNA),能折叠成特定的三维空间结构同靶物质进行高特异性与高亲和性的结合。近年来,以全细胞为靶标筛选(cell-SELEX)获得的核酸适配体在疾病相关的领域有很大的应用潜力,尤其在细胞分子的识别、生物标志物的发现等方面,但cell-SELEX的过程复杂、难度大及获得的核酸适配体性能不佳等问题仍然制约着细胞特异性核酸适配体的进一步发展,如何高效地筛选获得核酸适配体是其应用的关键。本文总结了目前在cell-SELEX技术基础上发展起来的新方法、新策略及核酸适配体在肿瘤研究中的应用,望为相关科研人员的研究提供参考。     

核酸适配体在三阴性乳腺癌诊疗中的研究进展*

乳腺癌是女性高发恶性肿瘤,三阴性乳腺癌(triple-negative breast cancer, TNBC)恶性程度极高,且发病机制复杂,是乳腺癌分型中预后最差的类型,但目前其早期筛查和诊断的敏感度仍处在较低水平。因此,亟须通过应用具有高度特异性的肿瘤标志物分子探针,实现其早期诊断和治疗。核酸适配体是在人工合成的随机单链核酸序列文库中,通过指数富集的配体系统进化技术(systematic evolution of ligands by exponential enrichment, SELEX)筛选获得的寡核苷酸序列。高效的分子识别能力使其成为最具潜力的生物靶向分子,在肿瘤诊断及治疗中具有广阔的应用前景。目前,通过筛选已获得了多种靶向TNBC细胞的核酸适配体。重点综述基于SELEX及其衍生技术筛选TNBC相关核酸适配体的新进展,以及核酸适配体在TNBC诊断和治疗中的应用,为相关研究提供参考。     

核酸适配体的体外筛选方法的最新研究进展

核酸适配体是用配体指数富集系统进化技术(SELEX)在体外筛选得到的一小段寡核苷酸序列,能够选择性的与不同的靶标特异性的结合,包括蛋白质、小分子、有机物、金属离子、药物等,具有高亲和力和高特异性。这项技术的诸多优势,使其迅速得到重视,核酸适配体在生物传感器、基因芯片、新药开发、纳米技术等诸多方面应用广泛。但是传统的SELEX方法操作繁琐,筛选周期长,需要几个月的时间才能筛选出与靶标具有高特异性的核酸适配体。随着SELEX的快速发展,近年来出现了很多新型的筛选方法,这些新的方法大大提高了筛选周期,极大的提高了筛选效率,拓展了核酸适配体的应用。总结介绍了近三年来出现的几种新型的核酸适配体的筛选方法,包括氧化石墨烯SELEX(Multiple GO-SELEX)、单壁碳纳米管辅助细胞SELEX(SWCNTs-assisted cell-SELEX)、基于芯片的细胞SELEX(on-chip Cell-SELEX)、序列构造SELEX(Sequence-constructive SELEX)、高保真SELEX(High-Fidelity SELEX),有助于人们进一步了解、认识核酸适配体筛选技术的发展现状,更好促进核酸适体在各个领域中的应用前景。     

以完整细胞为靶子的SELEX技术研究进展

指数富集的配体系统进化（SELEX）是一种从大容量寡核苷酸文库中经反复分离扩增步骤得到针对靶分子的高亲和力、高特异性核酸配基——适配体的体外筛选技术。自1990年以来,SELEX技术得到了迅猛发展,筛选的靶分子已由最初的单一物质发展到完整的动物细胞、细菌病原体等复杂靶子。以完整细胞为靶子的SELEX技术有其独特的技术优势,可以在筛选细胞上特定靶分子未知的情况下进行筛选,为药物筛选、临床诊断、疾病治疗和基础医学研究等带来了新的思路和方法。随着对适配体研究的深入,尤其是纳米材料与其相结合应用,该技术将在肿瘤诊断治疗及微生物检测领域具有更为广泛的应用前景。     

适配体的筛选及在生命分析化学中的应用

指数级富集的配体系统进化技术（SELEX）是近年来发展的获得能够与靶分子高特异性和高亲力结合的寡核苷酸序列（适配体）的筛选技术。目前多种靶分子的适配体如蛋白或小分子,都已经通过SELEX技术筛选获得,使适配体在蛋白质组研究、临床医学、药物研发及基因调控等领域已经成为重要的研究工具。本文就近几年适配体的筛选技术及在生命分析化学中的应用发展方面进行了综述。     

人FcγRI特异性核酸适配体的体外筛选及鉴定北大核心CSCD

人中性粒细胞FcγRI(即CD64),Ig G Fc高亲和力受体之一,是早期诊断脓毒血症和系统性细菌感染的一个灵敏和特异的新标志物;目前,采用流式细胞计量术测定,难以在一般实验室开展。本研究旨在应用新型的体外筛选技术——指数富集配基的系统进化(systematic evolution of ligands by exponential enrichment,SELEX)技术——从体外合成的随机寡核苷酸文库中筛选人FcγRI的高亲和力和高特异性的核酸适配体(aptamer)。本文以人FcγRI为靶标,将其固定在羧基活化磁珠上,进行SELEX筛选。经过8轮筛选,共获得3个重点研究的单克隆适配体。生物信息学分析结果显示,人FcγRI适配体的模拟二级结构以茎-环和G-四聚体为主,可能是FcγRI与适配体的作用位点,G-T错配常见。流式细胞计量术和荧光显微镜分析显示,筛选出的典型单克隆适配体解离常数(the dissociation constant,Kd)均达到纳摩尔水平,而且适配体只与脓毒血症中性粒细胞结合,具有良好的亲和力和特异性。本研究表明,通过SELEX技术,成功获取了人FcγRI特异性核酸适配体,为以此适配体为分子探针,进一步建立用于脓毒血症早期诊断的新方法奠定了基础。     

Characterisation of aptamer–target interactions by branched selection and high-throughput sequencing of SELEX pools

Nucleic acid aptamer selection by systematic evolution of ligands by exponential enrichment (SELEX) has shown great promise for use in the development of research tools, therapeutics and diagnostics. Typically, aptamers are identified from libraries containing up to 10¹⁶ different RNA or DNA sequences by 5–10 rounds of affinity selection towards a target of interest. Such library screenings can result in complex pools of many target-binding aptamers. New high-throughput sequencing techniques may potentially revolutionise aptamer selection by allowing quantitative assessment of the dynamic changes in the pool composition during the SELEX process and by facilitating large-scale post-SELEX characterisation. In the present study, we demonstrate how high-throughput sequencing of SELEX pools, before and after a single round of branched selection for binding to different target variants, can provide detailed information about aptamer binding sites, preferences for specific target conformations, and functional effects of the aptamers. The procedure was applied on a diverse pool of 2′-fluoropyrimidine-modified RNA enriched for aptamers specific for the serpin plasminogen activator inhibitor-1 (PAI-1) through five rounds of standard selection. The results demonstrate that it is possible to perform large-scale detailed characterisation of aptamer sequences directly in the complex pools obtained from library selection methods, thus without the need to produce individual aptamers.     

Simultaneous generation of aptamers to multiple gamma-carboxyglutamic acid proteins from a focused aptamer library using DeSELEX and convergent selection

By using the in vitro selection method SELEX against the complex mixture of GLA proteins and utilizing methods to deconvolute the resulting ligands, we were able to successfully generate 2'-ribo purine, 2'-fluoro pyrimidine aptamers to various individual targets in the GLA protein proteome that ranged in concentration from 10 nM to 1.4 microM in plasma. Perhaps not unexpectedly, the majority of the aptamers isolated following SELEX bind the most abundant protein in the mixture, prothrombin (FII), with high affinity. We show that by deselecting the dominant prothrombin aptamer the selection can be redirected. By using this DeSELEX approach, we were able to shift the selection toward other sequences and to less abundant protein targets and obtained an aptamer to Factor IX (FIX). We also demonstrate that by using an RNA library that is focused around a proteome, purified protein targets can then be used to rapidly generate aptamers to the protein targets that are rare in the initial mixture such as Factor VII (FVII) and Factor X (FX). Moreover, for all four proteins targeted (FII, FVII, FIX, and FX), aptamers were identified that could inhibit the individual protein's activitity in coagulation assays. Thus, by applying the concepts of DeSELEX and focused library selection, aptamers specific for any protein in a particular proteome can theoretically be generated, even when the proteins in the mixture are present at very different concentrations.     

Emulsion PCR: a high efficient way of PCR amplification of random DNA libraries in aptamer selection

Aptamers are short RNA or DNA oligonucleotides which can bind with different targets. Typically, they are selected from a large number of random DNA sequence libraries. The main strategy to obtain aptamers is systematic evolution of ligands by exponential enrichment (SELEX). Low efficiency is one of the limitations for conventional PCR amplification of random DNA sequence library in aptamer selection because of relative low products and high by-products formation efficiency. Here, we developed emulsion PCR for aptamer selection. With this method, the by-products formation decreased tremendously to an undetectable level, while the products formation increased significantly. Our results indicated that by-products in conventional PCR amplification were from primer-product and product-product hybridization. In emulsion PCR, we can completely avoid the product-product hybridization and avoid the most of primer-product hybridization if the conditions were optimized. In addition, it also showed that the molecule ratio of template to compartment was crucial to by-product formation efficiency in emulsion PCR amplification. Furthermore, the concentration of the Taq DNA polymerase in the emulsion PCR mixture had a significant impact on product formation efficiency. So, the results of our study indicated that emulsion PCR could improve the efficiency of SELEX.     

Advances in SELEX and application of aptamers in the central nervous system

SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is a screening technique that involves the progressive selection of highly specific ligands by repeated rounds of partition and amplification from a large combinatorial nucleic acid library. The products of the selection are called aptamers, which are short single stranded DNA or RNA molecules, binding with high affinity, attributed to their specific three-dimensional shapes, to a large variety of targets, ranging from small molecules to complex mixtures. Various improvement of the original SELEX method described in 1990 have been obtained recently, such as capillary electrophoresis SELEX, Toggle-SELEX, Tailored-SELEX, Photo-SELEX, and others. These new variants greatly shorten time of selection and improve aptamer affinity and specificity. Such aptamers have great potential as detecting and/or diagnostic reagents. Furthermore, some aptamers specifically inhibit biological functions of targeted proteins, and are considered as potent therapeutic lead structures evaluated in preclinical disease models. Recently, one aptamer has been approved by Food and Drug Administration of US for treating age-related macular degeneration. This review presents recent advances in the field of SELEX with special emphasis on applications of aptamers as analytical, diagnostic and therapeutic tools in the central nervous system.     

化学修饰寡核苷酸在核酸配基扩增技术中的应用

核酸酸基扩增技术（SELEX)可从极大容量的随机寡核苷酸文库中筛选得到与靶分子高特异性和高亲和力结合的核酸配基。对寡核苷酸进行化学修饰,可以提高核酸配基的稳定性,增加其功能多样性及生物利用度。SELEX在基础研究、诊断和治疗试剂的研制及药物筛选等领域有广泛用途。     

金黄色葡萄球菌外毒素B特异性适体的筛选及其应用

目的:利用指数富集配基的系统进化(SELEX)技术,筛选能与金黄色葡萄球菌外毒素B(SEB)特异、高亲和力结合的单链DNA(ssDNA)适体,并将该适体应用于患者血清标本的检测。方法:从体外合成的96核苷酸随机ss-DNA文库中,以羧基磁珠作为筛选介质,经逐步PCR扩增、筛选,获得针对SEB的高亲和力、高特异性适体;利用荧光素标记适体测定筛选过程中各轮结合力;利用酶连接适体方法检测适体特异性和结合力。结果:经过13轮筛选,ssDNA文库与SEB的结合百分率从1.1%提高到39.8%,增加了36倍;获得的ssDNA适体(A11)针对SEB的特异性强,与金黄色葡萄球菌表面蛋白A(SPA)结合低,并能初步识别患者血清。结论:利用SELEX技术筛选获得了特异结合SEB的高亲和力的ssDNA适体,为金黄色葡萄球菌的临床诊断与治疗奠定了基础。     

Three decades of nucleic acid aptamer technologies: Lessons learned,progress and opportunities on aptamer development

Aptamers are short single-stranded nucleic acid sequences capable of binding to target molecules in a way similar to antibodies. Due to various advantages such as prolonged shelf life, low batch to batch variation, low/no immunogenicity, freedom to incorporate chemical modification for enhanced stability and targeting capacity, aptamers quickly found their potential in diverse applications ranging from therapy, drug delivery, diagnosis, and functional genomics to bio-sensing. Aptamers are generated by a process called SELEX. However, the current overall success rate of SELEX is far from being satisfactory, and still presents a major obstacle for aptamer-based research and application. The need for an efficient selection strategy consisting of defined procedures to deal with a wide variety of targets is significantly important. In this work, by analyzing key aspects of SELEX including initial library design, target preparation, PCR optimization, and single strand DNA separation, we provide a comprehensive analysis of individual steps to facilitate researchers intending to develop personalized protocols to address many of the obstacles in SELEX. In addition, this review provides suggestions and opinions for future aptamer development procedures to address the concerns on key SELEX steps, and post-SELEX modifications.     

全细菌SELEX法筛选粪肠球菌特异性适配体

肠球菌（Enterococcus）是内源性和外源性医院感染的第二大病原菌,检出率仅次于大肠杆菌,从分子水平上发展靶标的高亲和力分子探针对肠球菌的识别和检测具有非常重要的意义。本研究以粪肠球菌为靶标,运用全细菌指数富集的配体系统进化技术（whole-bacteria systematic evolution of ligands by exponential enrichment, whole-bacteria SELEX）,从全长为79个核苷酸包含35个随机碱基序列的单链DNA文库中筛选与靶标高亲和力、高特异性结合的适配体,利用荧光分析法监控筛选过程中不同轮次所得次级文库与粪肠球菌的结合力,经12轮筛选和克隆测序,获得了39条适配体序列。进一步对筛选得到的适配体进行序列比对、二级结构分析、流式细胞分析、解离常数（Kd）测定及特异性验证,最终获得一条与粪肠球菌能特异性结合的适配体Apt 21,其Kd值为549.2 ± 147.4 nmol/L。该适配体可作为粪肠球菌检测的识别元件,为建立基于适配体的新型粪肠球菌检测方法奠定了基础。