抗体Fc片段特异寡核苷酸配基介导的实时定量免疫PCR

目的:利用小鼠IgG抗体Fc片段高特异、高亲和寡核苷酸配基,构建实时定量免疫PCR检测方法,提高抗体检测的灵敏度。方法:用SELEX技术从随机寡核苷酸文库中筛选抗体Fc片段特异寡核苷酸配基,设计合成信标序列,通过不对称PCR法,制备IgG Fc片段的核酸信标配基分子;32P标记核酸信标配基,采用琼脂糖凝胶阻滞双显色法鉴定核酸信标配基与IgG Fc片段结合的亲和力和特异性;制备IgG Fc特异性寡核苷酸信标配基-抗体复合检测分子,构建小鼠IgG Fc片段特异核酸信标配基介导的实时定量免疫PCR检测方法。结果:制备了IgG Fc片段的核酸信标配基分子;凝胶阻滞放射自显影和考马斯亮蓝二次染色结果显示该核酸信标配基分子与IgG Fc片段具有高度亲和力和活性,而且只与非变性IgG结合,与变性IgG不结合;IgG Fc片段的特异核酸信标配基与IgG结合形成复合检测分子,有效完成了信号传递和实时定量PCR信号放大过程。结论:初步建立了一种全新的核酸信标配基介导的免疫PCR检测方法,可有效提高现有IgG类抗体免疫检测的灵敏度和特异性。     

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。     

SELEX技术及Aptamer研究的新进展

综述了近几年指数富集的配体系统进化(SELEX)技术的改良与寡核苷酸配基(aptamer)研究应用方面的新进展.Aptamer是指利用SELEX(systematicevolutionofligandsbyexponentialenrichment)技术,从随机寡核苷酸文库中筛选获得的能够与靶分子特异结合的短单链寡核苷酸配基,通常具有纳摩尔到皮摩尔的亲和力.高通量筛选的技术特点与aptamer精确识别、易体外合成与修饰等特性,使得aptamer在分析化学与生物医药研究方面具有广阔的应用前景.     

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。     

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。     

应用双向热循环消减SELEX技术筛选肺癌血清核酸适配体

肺癌是发病率和死亡率较高的恶性肿瘤。现阶段,用于肺癌早期诊断的血清肿瘤标志物因其特异性与敏感性均较低,严重影响肺癌的临床诊断和治疗。本文用双向热循环消减指数富集的配基进化(systematic evolution of ligands by exponential enrichment, SELEX)技术,筛选肺癌和非癌血清标志物的核酸适配体,建立肺癌的检测方法,提高诊断和治疗效率。实验用环氧基琼脂磁珠为筛选介质,以非癌混合血清、肺癌混合血清作为双向靶标。应用热循环消减SELEX技术,经19轮筛选分别获得非癌和肺癌血清的特异性核酸适配体。通过高通量测序,得到 40条非癌核酸适配体序列和 41条肺癌核酸适配体序列。从肺癌与非癌血清特异性核酸适配体序列中分别挑选出高丰度的 4条序列,合成后制成检测试剂,经临床血清验证,阳性率为 90%。该检测方法检测灵敏度高,为肺癌的早期诊断和治疗提供了新的分子识别元件。     

指数富集配基的系统进化原理及应用进展

过去十几年,指数富集配基的系统进化（SELEX）技术得到了充分的发展,目前已经成为在数量众多的靶分子中,高亲和性和特异性地确定适配子的一种崭新的方法.SELEX的流程即是人工合成随机寡核苷酸序列、适配子筛选、扩增、再循环的过程.在这一过程中,适配子修饰基团文库的应用使得SELEX流程更加经济、快捷、高通量.回顾了SELEX的研究和应用,期待着这一方法能够在理论和实践上得到进一步的发展.     

核酸适配体结合纳米材料用于肿瘤靶向治疗

核酸适配体(aptamer)是一类由指数富集配体系统进化(systematic evolution of ligands by exponential enrichment,SELEX)技术筛选出的RNA和单链DNA寡核苷酸片段。因其非同寻常的分子识别能力及结构特性,核酸适配体已经成为最具有应用前景的生物分子之一,综述了核酸适配体结合不同的纳米材料在肿瘤靶向治疗上的应用。     

应用双向热循环消减SELEX技术筛选胃癌血清核酸适配体

胃癌是发病率及死亡率均较高的消化道恶性肿瘤之一,严重威胁人类的生命健康。血清肿瘤标志物的检测对提高早期胃癌的检出率,改善胃癌的治疗有重要的意义。核酸适配体以其灵敏度高、靶向性强等优势显示出了较强的临床适用性。本研究以双向热循环消减指数富集式配基系统进化(systematic evolution of ligands by exponential enrichment,SELEX)技术为支持,纳米（琼脂）磁珠材料为载体,胃癌血清及正常人血清为筛选靶标,结合高通量测序技术建立了快速高效的核酸适配体筛选方法。经过19轮双向筛选,获得高重复率的胃癌血清特异性核酸适配体序列10条及正常人血清特异性核酸适配体序列8条。将这些序列分别混合并制成检测试剂A、B,结合实时荧光定量PCR（quantitative real-time PCR,qPCR）技术,对100份临床血清样本进行特异性验证。通过比较分析,建立了快速高效的胃癌血清检测技术。结果显示,核酸适配体G AP1与N AP1的二级结构类似于抗体的“Y”型,且呈茎环状。检测试剂A、B对胃癌及正常人血清的检出率分别为92%和88%。表明本技术可以较准确地筛选得到高特异性和强亲和力的核酸适配体,体现了核酸适配体作为新型肿瘤标志物在临床检测及治疗的应用潜力。     

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。     

Combinatorial Chemistry of Nucleic Acids: SELEX

A method of irrational oligonucleotide design, SELEX, is considered. Individual SELEX products, aptamers, are small molecules (40–100 nt) that have a unique three-dimensional structure, which provides for their specific and high-affinity binding to targets varying from low-molecular-weight ligands to proteins. Thus, the sophisticated biosynthesis of recognizing protein elements, antibodies, can be emulated in vitro via selection and synthesis of principally new recognizing elements based on nucleic acids.     

Assays for cytokines using aptamers

Aptamers are short nucleic acid sequences that are used as ligands to bind their targets with high affinity. They are generated via the combinatorial chemistry procedure systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have shown much promise towards detection of a variety of protein targets, including cytokines. Specifically, for the determination of cytokines and growth factors, several assays making use of aptamers have been developed, including aptamer-based enzyme-linked immunosorbent assays, antibody-linked oligonucleotide assay, fluorescence (anisotropy and resonance energy transfer) assays, and proximity ligation assays. In this article, the concept of aptamer selection using SELEX and the assay formats using aptamers for the detection of cytokines are discussed.     

Binding of an aptamer to the N-terminal fragment of VCAM-1

In vitro selection of 2'-fluoropyrimidine oligonucleotide aptamers was performed against the N-terminal two-domain fragment of mouse VCAM-1. The SELEX procedure enriched the starting pool in a family of homologous sequences. High binding affinity (10nM) of one member of this family, aptamer 12.11, was demonstrated in a filter binding assay.     

In vitro selection using a dual RNA library that allows primerless selection

High affinity target-binding aptamers are identified from random oligonucleotide libraries by an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). Since the SELEX process includes a PCR amplification step the randomized region of the oligonucleotide libraries need to be flanked by two fixed primer binding sequences. These primer binding sites are often difficult to truncate because they may be necessary to maintain the structure of the aptamer or may even be part of the target binding motif. We designed a novel type of RNA library that carries fixed sequences which constrain the oligonucleotides into a partly double-stranded structure, thereby minimizing the risk that the primer binding sequences become part of the target-binding motif. Moreover, the specific design of the library including the use of tandem RNA Polymerase promoters allows the selection of oligonucleotides without any primer binding sequences. The library was used to select aptamers to the mirror-image peptide of ghrelin. Ghrelin is a potent stimulator of growth-hormone release and food intake. After selection, the identified aptamer sequences were directly synthesized in their mirror-image configuration. The final 44 nt-Spiegelmer, named NOX-B11-3, blocks ghrelin action in a cell culture assay displaying an IC₅₀ of 4.5 nM at 37°C.     

An in vitro-selected RNA-binding site for the KH domain protein PSI acts as a splicing inhibitor element

P element somatic inhibitor (PSI) is a 97-kDa RNA-binding protein with four KH motifs that is involved in the inhibition of splicing of the Drosophila P element third intron (IVS3) in somatic cells. PSI interacts with a negative regulatory element in the IVS3 5' exon. This element contains two pseudo-5' splice sites, termed F1 and F2. To identify high affinity binding sites for the PSI protein, in vitro selection (SELEX) was performed using a random RNA oligonucleotide pool. Alignment of high affinity PSI-binding RNAs revealed a degenerate consensus sequence consisting of a short core motif of CUU flanked by alternative purines and pyrimidines. Interestingly, this sequence resembles the F2 pseudo-5' splice site in the P element negative regulatory element. Additionally, a negative in vitro selection of PCR-mutagenized P element 5' exon regulatory element RNAs identified two U residues in the F1 and F2 pseudo-5' splice sites as important nucleotides for PSI binding and the U residue in the F2 region is a nearly invariant nucleotide in the consensus SELEX motif. The high affinity PSI SELEX sequence acted as a splicing inhibitor when placed in the context of a P element splicing pre-mRNA in vitro. Data from in vitro splicing assays, UV crosslinking and RNA-binding competition experiments indicates a strong correlation between the binding affinities of PSI for the SELEX sequences and their ability to modulate splicing of P element IVS3 in vitro.     

Neutralizing aptamers from whole-cell SELEX inhibit the RET receptor tyrosine kinase

Targeting large transmembrane molecules, including receptor tyrosine kinases, is a major pharmacological challenge. Specific oligonucleotide ligands (aptamers) can be generated for a variety of targets through the iterative evolution of a random pool of sequences (SELEX). Nuclease-resistant aptamers that recognize the human receptor tyrosine kinase RET were obtained using RET-expressing cells as targets in a modified SELEX procedure. Remarkably, one of these aptamers blocked RET-dependent intracellular signaling pathways by interfering with receptor dimerization when the latter was induced by the physiological ligand or by an activating mutation. This strategy is generally applicable to transmembrane receptors and opens the way to targeting other members of this class of proteins that are of major biomedical importance.     

Aptamer selection for the detection of Escherichia coli K88

In this study, the first group of single-stranded DNA aptamers that are highly specific to enterotoxigenic Escherichia coli (ETEC) K88 was obtained from an enriched oligonucleotide pool by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, during which the K88 fimbriae protein was used as the target and bovine serum albumin as counter targets. These aptamers were applied successfully in the detection of ETEC K88. They were then grouped under different families based on the similarity of their secondary structure and the homology of their primary sequence. Four sequences from different families were deliberately chosen for further characterization by fluorescence analysis. Having the advantage of high sensitivity, fluorescence photometry was selected as single-stranded DNA quantification method during the SELEX process. Aptamers with the highest specificity and affinity were analyzed to evaluate binding ability with E. coli. Since ETEC K88 is the only type of bacterium that expressed abundant K88 fimbriae, the selected aptamers against the K88 fimbriae protein were able to specifically identify ETEC K88 among other bacteria. This method of detecting ETEC K88 by aptamers can also be applied to bacteria other than ETEC K88.     

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。