成纤维细胞生长因子受体1配体精细结合位点的确定

目的 确定人成纤细胞生长因子受体1（FGFR1）的配体精细结合位点。方法 合成肽文库筛选，定向点突变,源核细胞重组蛋白质表达及受体－配体结合实验。结果 采用^125I标记的酸性成纤维细胞生生长因子（aFGF)，自合成肽文库中筛选到一个五肽序列（WGPGM），其序列及立体结构和FGFR1细胞外段的一个基序（WISPEKM）相似。为了证明FGFR1的WTSPEKM基序是结合FGF的重要结合，采用定向突变技术将WITSPEKM基序中的P（CCA）突变成A（GCA），并在原核细胞中表达了野生型和突变型FGFR1细胞外段重组蛋白质，受体－配体结合实验显示突变的GFFR1细胞外段结合aFGF的能力明显降低。结论 FGFR1的WISPEKM基序是配体结合的一个重要部位。     

SELEX技术及其应用研究进展

SELEX技术是一类在体外筛选能与各种配体特异结合的寡聚核苷酸片段的一项新组合化学技术，具有靶分子范围广，筛选出的配体亲和力和特异性高等特点。筛选出的特异寡聚核苷酸片段，不仅在疾病的诊断、治疗与药物的快速开发等方面起着重要作用，也为核酸结构和功能的研究提供了一个有效的方法。     

HIV-p24核酸适配体的筛选

 摘 要：目的 利用SELEX技术筛选HIV-p24的核酸适配体,为艾滋病的诊断和治疗奠定基础。方法 以重组p24为筛选靶,用SELEX技术从随机寡核苷酸库中筛选与HIV-p24结合的寡核苷酸,利用凝胶阻滞实验鉴定第12轮筛选到的寡核苷酸与HIV-p24的结合,再用Dot-blot法筛选出与HIV-p24结合的核酸适配体,并检测核酸适配体识别HIV-p24的特异性。结果 Dot-blot筛选到5条与HIV-p24有较强结合能力的核酸适配体,且均为不同的序列。特异性检测显示,18和26号配体只与HIV-p24特异性结合,与人血清白蛋白、牛血清白蛋白和脱脂奶粉均无明显结合。结论 成功筛选到2条特异结合HIV-p24的核酸适配体,为其应用于艾滋病诊断和治疗提供了实验基础。     

SELEX技术及其应用研究进展

SELEX技术是一类在体外筛选能与各种配体特异结合的寡聚核苷酸片段的一项新组合化学技术,具有靶分子范围广,筛选出的配体亲和力和特异性高等特点.筛选出的特异寡聚核苷酸片段,不仅在疾病的诊断、治疗与药物的快速开发等方面起着重要作用,也为核酸结构和功能的研究提供了一个有效的方法.     

针对于白蛋白的新型核酸适配体的筛选与鉴定

目的研发能特异结合白蛋白的DNA核酸适配体,为去除实验样品中的白蛋白提供新型技术手段。方法体外合成全长为59个碱基并含有21个随机寡核苷酸的单链DNA文库,以白蛋白为靶标,利用指数富集的配体系统进化技术(SELEX),从单链DNA文库中筛选能特异结合白蛋白的核酸适配体;用流式细胞计量术来检测核酸适配体的富集程度、适配体与白蛋白的结合特性;用MFold软件预测其二级结构。结果经过多轮筛选得到了能特异识别白蛋白的DNA核酸适配体A6,其Kd值为77.4 nmol/L,且基本不结合卵清蛋白、免疫球蛋白Ig G等对照蛋白。结论针对于白蛋白的DNA核酸适配体A6能选择性识别白蛋白,在去除实验样品中的白蛋白方面具有应用潜能。     

基于细胞的指数富集配体系统进化技术的肺癌细胞核酸适配体的筛选及其在肿瘤诊治中的应用

核酸适配体是经过指数富集配体系统进化技术(SELEX)筛选得到的寡核苷酸序列。已有研究表明,核酸适配体在肿瘤诊断及治疗方面具有良好的应用前景。因此,本文主要针对肺癌细胞核酸适配体的筛选、表征等方面展开论述,初步探讨核酸适配体作为靶向载体和靶向药物在肿瘤诊断治疗中的作用,为肿瘤的早期诊断及早期治疗提供新思路。     

受体放射分析中标记配体质量的评价

受体的放射性配体结合分析是目前受体研究中常用的方法。其中，标记配体的质量十分重要。标记配体的质量主要有两个方面，即标记配体的纯度（结合力）及其与未标记配体的结合性能的一致性。目前，多数文献报道主雍以受体饱和结合试验的双倒数来测试标记配体的结合力，再以结合力校正标记配体量，得到活性的杯记配体量。在此基础上进行自身转换试验，测定标记配体比放射性及其与未标记配体结合性能的等效性。采用这些方法，作者对＾１     

Aptamer核酸药物的研究进展

Ａｐｔａｍｅｒ指的是能结合蛋白或其他小分子物质的单链或双链寡核苷酸〔1〕 。体外筛选技术的发展和ＰＣＲ技术的应用 ,使得Ａｐｔａｍｅｒ的研究近年来有了长足的进步 ,筛选到了一大批能与各种蛋白或小分子特异紧密结合的核酸分子(Ａｐｔａｍｅｒ)。这些Ａｐｔａｍｅｒ包含了ＲＮＡ、双链ＤＮＡ、单链ＤＮＡ等多种形式的寡核苷酸 ,其配体的性质各异。体外筛选Ａｐｔａｍｅｒ不仅增强了人们对核酸 蛋白相互作用的认识 ,也为寻找新药提供了一条途径。Ａｐｔａｍｅｒ核酸药物的研究近年来也取得了一些可喜成果。我们拟就该方面的进展及相关…     

用原子力显微技术测定生物分子之间的相互作用力

原子力显微技术（ＡＦＭ）可直接测定生物大分子之间的作用力，为人们理解生命原理提供了一个新的手段，目前已用ＡＦＭ对一些在生命科学中具有重要意义的生物大分子之间的任务和进行了测定如；（１）配体－受体之间的相互作用力：（２）运动蛋白之间的作用力；（３）细胞粘附糖蛋白之间的作用力；４）核酸双锭和碱基地     

针对干扰素-γ的新型核酸适配体的筛选及鉴定

 目的: 研发能特异性结合干扰素-γ（IFN-γ）的DNA适配体,为发展IFN-γ的新型检测技术提供基础。方法: 体外合成全长为59个碱基并含有21个随机寡核苷酸的单链DNA文库,以IFN-γ蛋白为靶标,利用指数富集的配体系统进化技术（SELEX）,从单链DNA文库中筛选能够选择性结合IFN-γ蛋白的核酸适配体;流式细胞术检测富集进度、适配体与IFN-γ蛋白结合特性;MFold软件预测二级结构。结果: 经多轮筛选获得了能够识别IFN-γ蛋白的DNA适配体B3-8,其能够选择性地结合IFN-γ蛋白,而与血清白蛋白的结合较弱。经检测其Kd值为185 nmol/L。结论: DNA适配体B3-8能选择性地识别IFN-γ蛋白,在研发针对IFN-γ的新型检测技术方面具有应用潜能。     

Application of Aptamers for Targeted Therapeutics

Aptamers are short, single-stranded oligonucleotides that are isolated through a process termed systematic evolution of ligands by exponential enrichment. With the advent of cell-based selection technology, aptamers can be selected to bind protein targets that are expressed on the cell surface. These aptamers demonstrate excellent specificity and high affinity toward their target proteins and are often internalized upon binding to their targets. This has opened up the possibility of using aptamers for cell-specific targeted drug delivery. In this review, we will discuss cell-surface protein targets, the aptamers that bind them, and their applications for targeted therapeutics.     

SELEX与适配体在蛋白质研究中的应用

指数富集的配基系统进化(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。SELEX技术自1990年发展至今,已涌现出多种筛选模式和分离方法。筛选特异结合蛋白质的SELEX技术发展直接影响和指导了适配体在蛋白质功能调控方面的应用。文章综述了多种SELEX技术在筛选蛋白质方面的发展近况,适配体在蛋白质功能研究中的应用,筛选过程中关键性因素的确定及适配体的前后期修饰。     

Dynamic evolution and immunoreactivity of aptamers binding to polyclonal antibodies against MPT64 antigen of Mycobacterium tuberculosis

Antibody responses can be useful markers of tuberculosis infection. However, the established immunoassay diagnostic method is limited by antigenic variability. Replacing the recombinant proteins with aptamers may overcome these antigenic challenges. In this study, we systematically monitored the selection process of aptamers against anti-MPT64 antibodies of Mycobacterium tuberculosis to obtain more aptamers for developing a multisite system to increase the sensitivity of TB serological diagnosis. Twelve high-affinity aptamers with distinctive secondary structures were obtained by analyzing the dynamic evolution of aptamers against anti-MPT64 antibodies in the process of system evolution of ligands by exponential enrichment (SELEX). Pocket and stem-loops were found to be the basis of these aptamers binding to antibodies. Point mutations of highly conserved nucleotides in the pocket and stem-loop structures resulted in decreased affinity of aptamers to targets. To test the potential of these aptamers for future use in a serological diagnostic tool, three high-affinity aptamers with different epitope specificities were applied as capture aptamer in an enzyme-linked immunosorbent assay (ELISA) with sera of TB patients. The results showed that three aptamers all effectively bound anti-MPT64 antibodies from TB patients and had high specificity and sensitivity. These aptamers with high immunoreactivity in human sera may represent an efficient and promising analogue of MPT64 and have potential to substitute MPT64 as a nucleic acid antigen in the serological diagnosis of TB. Moreover, these aptamers with different epitope specificities may facilitate the development of a sandwich assay platform or a multisite system to effectively capture more targets in sera.     

DC-SIGN特异性适配子的筛选及鉴定

目的:筛选高特异性、高亲合力结合DC-SIGN的单链DNA适配子,为开发抗HIV、HCV和结核杆菌感染的新型预防和治疗试剂奠定基础。方法:采用基于细胞表面展示的SELEX技术(TECS-SELEX),筛选出具有高亲合力结合DC-SIGN的单链DNA(ssDNA)适配子;运用流式细胞术(FCM)检测该适配子的亲合力;对最高亲合力适配子库进行克隆和测序;ELISA和FCM方法检测单适配子ZD8与DC-SIGN蛋白结合的剂量相关性;MTT法测定IC50观察适配子对细胞的毒性。结果:第14轮筛选的适配子库亲和力最高;DC-SIGN抗体能抑制适配子结合表达DC-SIGN蛋白的细胞;第14轮库中筛选的单适配子ZD8与DC-SIGN蛋白的结合率呈剂量依赖性;所筛选的单适配子对肝细胞几乎无毒性。结论:成功筛选出DC-SIGN蛋白的ssDNA适配子,为防治HIV、HCV和结核杆菌感染等DC-SIGN相关性疾病,提供新型预防和治疗的候选试剂和小分子药物。     

Nucleic acid aptamers in human viral disease

Nucleic acid aptamers are short, single-stranded oligonucleotides or their modified analogues which avidly and specifically interact with targeted ligands through their 3-dimensional structure. Aptamers can be selected out of a large combinatorial oligonucleotide library through an in vitro evolution process termed SELEX. Since 1990, a wide variety of aptamers targeted to ligands ranging from small molecules to complex mixtures have been isolated. Most selected aptamers have shown high specificity to and affinity for their ligands and are potential detection and/or diagnostic reagents. Furthermore, some aptamers specifically inhibit biological functions of targeted proteins, resulting in potent therapeutic candidates in disease models. Some recent advances to increase the stability of aptamers, extend their in vivo circulation time and their in vivo expression have pushed aptamers closer to therapeutic applications. This review presents recent developments in the field of aptamer research and focuses on their applications to human viral diseases, particularly HIV induced diseases.     

68A new way of targeting protease-receptor binding: Selection of uPA binding RNA aptamers inhibiting its association to its receptor uPAR

The binding of urokinase-type plasminogen activator (uPA) to its cell surface receptor (uPAR) has been implicated in tumour spread. We have employed a new method for developing inhibitors of the binding. Systematic evolution of ligands by exponential enrichment (SELEX) is an approach to selecting for RNA or DNA oligonucleotides with specific properties. Selections can be performed from a large pool of random sequences (10¹⁵) of RNA or DNA oligonucleotides which fold into three-dimensional structures. It is then possible to select RNA or DNA oligonucliotides binding with high affinities to targets of interest. These binding specific oligonucleotides are called aptamers. In a SELEX experiment, we used a library of serum-stable 2'-fluoro-pyrimidine modified RNA oligonucleotides to select for RNA aptamers capable of binding to urokinase-type plasminogen activator (uPA). Analysed by surface plasmon resonance (SPR), the selected aptamers bind to the amino terminal fragment of human uPA with K_D -values in the low nanomolar range. Cell binding assays showed that the selected aptamers were able to inhibit the binding of the amino terminal fragment of uPA to the receptor uPAR expressed on U937 cells. The same conclusion was reached by SPR analyse. The aptamers showed no inhibition of uPA's proteolytic activity. Based on computerised secondary structure predictions, it was possible to reduce size of the aptamers down to 49 nucleotides without losing the inhibitory properties. uPA-binding aptamers could be a promising tool for interfering with the pathophysiological functions of the plasminogen activation system. The inhibition of the uPA-uPAR interaction will inhibit plasminogen activation on the cells surface without interfering with the other function of uPAR and uPA. Aptamers are also interesting tools for analytic and imaging purpose because of the small size and high affinities.     

Aptamers: selection and scope of applications

Aptamers are short oligonucleotides selected from large combinatorial pools of sequences for their capacity to bind to many different targets ranging from small molecules (amino acids, antibiotics...) to proteins or nucleic acid structures. Aptamers present the same high specificity and affinity for their targets as antibodies. In addition to efficient binding, aptamers have been shown in many cases to display an inhibitory activity against their targets. Many aptamers are now being developed against biomedical relevant targets, and one aptamer that inhibits the human VEGF165 already received approval for the treatment of age-related macular degeneration. Here we discuss the principles and the practical way of selecting aptamers (SELEX technology) as well as the structural basis for their performance as ligands. A wide scope of applications is described - aptamers have been used as tools for studying nucleic acids/proteins interactions, detecting, purifying or imaging target molecules, regulating gene expression - and includes recent developments of aptamers for therapy and diagnosis.     

Cell adhesion on an artificial extracellular matrix using aptamer-functionalized PEG hydrogels

The development of an artificial extracellular matrix (ECM) is important to regenerative medicine because the ECM plays complex and dynamic roles in the regulation of cell behavior. In this study, nucleic acid aptamers were applied to functionalize hydrogels for mimicking the adhesion sites of the ECM. The results showed that nucleic acid aptamers could be incorporated into polyethylene glycol (PEG) hydrogels via free radical polymerization. The incorporation of the aptamers produced only a moderate effect on the mechanical properties of the PEG hydrogels. Importantly, the results also showed that the aptamers effectively induced cell type-specific adhesion to the PEG hydrogels without affecting cell viability. The cell adhesion was a function of the aptamer concentration, the spacer length and the cell seeding time. In addition, cell adhesion to the aptamer-functionalized hydrogel could be attenuated by means of aptamer inactivation in a physiological condition. Thus, aptamer-functionalized hydrogels are promising biomaterials for the development of artificial ECMs.     

DNA and RNA aptamers as modulators of protein function

The SELEX technique (systematic evolution of ligands by exponential enrichment) is a combinatorial library approach in which DNA or RNA molecules are selected by their ability to bind their protein targets with high affinity and specificity. The isolated molecules are referred to as aptamers (from aptus = Latin "to fit"). First, RNA and DNA aptamers were identified that bind to proteins naturally interacting with nucleic acids, or to small organic molecules such as ATP. In the following years, the use of the SELEX technique was extended to isolate oligonucleotide ligands for a wide range of proteins of importance for therapy, and diagnostics. Since these RNA and DNA molecules bind their targets with similar affinities as antibodies, and are able to distinguish between isotypes of an enzyme, aptamers have been also called synthetic antibodies. Recently, the use of in vitro selection methods to isolate protein inhibitors has been extended to complex targets, such as receptors that are only functional in their membrane-bound form, cells, and trypanosomes. RNA aptamers have been expressed in living cells where they inhibit a protein implicated in intracellular signal transduction. The utility of aptamers for in vivo experiments, and diagnostic and therapeutic purposes, is considerably enhanced by introducing chemical modifications into the oligonucleotides to provide resistance against enzymatic degradation in body fluids. Recently, such inhibitors have been evolved for a great variety of targets, including receptors, growth factors, and adhesion molecules implicated in disease. Furthermore, some results were already obtained in animal models and clinical trials.