SELEX技术及其在医学中的应用

指数富集配基的系统进化(SELEX)技术是一类新的组合化学技术,应用人工合成的随机寡核苷酸文库,通过筛选、分离、富集获得能与各种配体特异结合的寡核苷酸适配子(Aptamer),且具有高亲和力。目前涉及多种疾病治疗的适配子,包括感染性疾病,肿瘤,心血管疾病,免疫系统疾病等已被筛选出来,并且疗效已经通过体外实验或动物模型得到证实。两个适配子已进入药物III期临床实验阶段,还有很多适配子正处于预临床实验阶段,其中部分将很快进入临床实验。本文综述了SELEX技术及寡核苷酸适配子在治疗领域的最新进展,并且对其前景进行了分析和预测。     

铜绿假单胞菌表面脂多糖适配体的筛选及其抑制巨噬细胞极化的研究北大核心CSCD

目的筛选与铜绿假单胞菌（Pseudomonas aeruginosa,PA）表面脂多糖（LPS）特异性结合的适配体,探讨其抑制巨噬细胞极化的效果。方法提取PA的LPS成分,通过指数富集的配基系统进化技术（SELEX）筛选与其特异性结合的高亲和力DNA适配体,利用酶联寡聚核苷酸吸附试验（ELONA）、定量PCR（Q-PCR）等方法探讨其对巨噬细胞极化的影响。结果本研究筛选出能与PA-LPS特异性结合的适配体PL-6,并发现其能够阻断PA-LPS与相应受体TLR4的结合,抑制巨噬细胞M1型过度极化,维持巨噬细胞稳态。结论本研究筛选出可与PA-LPS特异性结合的高亲和力适配体,为PA可能引起脓毒症的预防和治疗提供了新的策略。     

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。     

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技术的原理和核酸适配体在医学上的应用进展

SELEX是一种制备能与靶分子高特异性高亲和力结合的寡核苷酸的技术,所制备的寡核苷酸分子被称为适配体(aptamer)。适配体可以是单链或双链DNA或RNA,是从非常大的随机的DNA或RNA寡核苷酸库中,经过多个循环的筛选和富集而获得的;它们的特点是能形成一定的立体结构,通过空间构象的密切适应而与靶分子高特异性、高亲和力地结合。适配体可用于医学和生命科学等领域的研究和疾病的诊断与治疗;适配体最大的优点是没有免疫原性,故在临床治疗时能重复使用;目前已有几种适配体进入了临床应用或临床试验。     

核酸适配体在肿瘤血清标志物筛选中的应用研究

肿瘤血清标志物的核酸适配体是利用指数富集的系统进化技术(SELEX),从体外筛选获得的一种能与肿瘤血清标志物高特异性、高亲和性结合的寡核苷酸分子。利用核酸适配体的优势,探寻对肿瘤更具诊断价值的新的肿瘤标志物。目前在肝癌、胃癌等肿瘤的研究中已经取得了一定成果。本文综述了当前核酸适配体及其在肿瘤血清标志物筛选中的相关研究,讨论了肿瘤血清标志物的核酸适配体在肿瘤诊断中的价值以及目前的研究所存在的问题。期望通过本文的综合描述,有助于人们更好地了解肿瘤血清标志物核酸适配体的筛选,并为新的肿瘤血清标志物的发现提供帮助。     

核酸适配体在肿瘤学中的应用研究进展

核酸适配体是一类通过指数级富集的配体进化技术(SELEX)获得的能以较高的亲和力与各类生物靶标特异性相结合的单链DNA或RNA。利用细胞-SELEX(Cell-SELEX)技术筛选出来的核酸适配体越来越多的作为靶向分子应用在肿瘤细胞检测、靶向药物载体和siRNA的研究中。体内外研究证明,核酸适配体作为靶向分子能用于肿瘤细胞的体外检测和肿瘤组织的体内成像,并能显著提高药物或siRNA对肿瘤细胞的杀伤作用。     

Progress in aptamers for cancer research

核酸适配体是一类通过指数级富集的配体进化技术(SELEX)获得的能以较高的亲和力与各类生物靶标特异性相结合的单链DNA或RNA.利用细胞-SELEX (Cell-SELEX)技术筛选出来的核酸适配体越来越多的作为靶向分子应用在肿瘤细胞检测、靶向药物载体和siRNA的研究中.体内外研究证明,核酸适配体作为靶向分子能用于肿瘤细胞的体外检测和肿瘤组织的体内成像,并能显著提高药物或siRNA对肿瘤细胞的杀伤作用.     

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。     

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。     

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.     

SELEX技术筛选结核分枝杆菌MPT64蛋白DNA适配子方法学研究

目的 利用指数富集配体系统进化(SELEX)技术筛选能与结核分枝杆菌MPT64蛋白特异结合的寡核苷酸适配子.方法 体外合成长度为78个核苷酸的随机ssDNA文库,利用SEL-EX技术筛选,以MPT64蛋白为靶物质进行12轮筛选,利用生物素-亲和素显色系统检测寡核苷酸与蛋白的结合性.结果 SELEX技术筛选MPT64蛋白适配子的技术体系:PCR扩增的最佳退火温度为65℃;文库优化时,最佳Mg2+的浓度为1.5 mmol/L;不对称PCR法制备ssDNA时,Mg2+的浓度为0.75 mmol/L;以酶联板为介质筛选时,在10轮后筛选达到饱和,且随着筛选轮数的增加,PCR扩增产物的电泳条带逐渐单一、致密,亲和性检测显示第10轮获得的适配子库,比初始的文库亲和性吸光度(A)值增加了9.18倍.将适配子库克隆,随机挑取10个单克隆子,利用混合夹心法检测其与MPT64的亲和性,亲和性范围分布在0.572～1.606之间.结论 已初步筛选到与MPT64蛋白高亲和性结合的DNA适配子.     

丙型肝炎病毒核心蛋白寡核苷酸适配子的筛选与鉴定

目的：筛选、鉴定抗HCV核心蛋白（C蛋白）的寡核苷酸适配子（aptamers）。方法：利用systematic evolution of ligands by exponential enrichment(SELEX)技术，以HCV C蛋白为靶分子，从体外合成的81bp随机单链DNA文库中筛选与HCV C蛋白特异结合的寡核苷酸适配子，并进行了解离常数（Kd）测定和适配子序列测定。再分别利用Clustal W软件包和DNA Folding Sever分析适配子的一级结构和二级结构。结果：经过9轮循环筛选，随机ssDNA库与HCV C蛋白的结合率从0.5%上升到32.5%。所有的一级结构没有共同的同源序列，但可分5个家族，每个家族具有共同的保守序列。二级结构分析表明，适配子形成的茎环、凸环结构可能是与HCV C蛋白结合的结构基础。其中寡核苷酸适配子C4与HCV C蛋白特异结合的亲和力最高，Kd值为68nmol/L。结论：利用随机寡核苷酸文库成功获得抗HCV C蛋白的寡核苷酸适配子。     

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.     

核酸适体技术研究进展

核酸适体（aptamer）指的是经体外筛选技术SELEX（指数富集配体系统进化）筛选出的能特异结合蛋白质或其他小分子物质的寡聚核苷酸片段，对可结合的配体有严格的识别能力和高度的亲和力。核酸适体在生物传感器、新药开发以及纳米技术等方面有着广泛的用途，本文对核酸适体近两年的研究进展进行综述。     

Subtractive SELEX against two heterogeneous target samples: numerical simulations and analysis

Systematic evolution of ligands by exponential (SELEX) is a revolutionary technology that integrates combinatorial chemistry with high throughput screening to generate from synthesized nucleic acid ligand libraries the high affinity nucleic acid ligands (aptamers) for interesting targets. Recently, the SELEX experiments have advanced from targeting the ligand libraries by a single purified target to multiple heterogeneous target samples. Having the potential of bringing enormous technical and economical advantages to drug discovery, the new application suffers from unpredictable performances. To gain an insight of the new method, we develop a computer model to numerically analyze the subtractive SELEX alternatively against two distinct heterogeneous samples of unknown targets. The model features the discretization of ligand library, the ligand-target binding equilibrium equations, and the separation efficiency of bound and unbound ligands in experiments. By computer simulations, we investigate how aptamers for desired targets embedded in undefined target mixtures are generated under different experimental conditions. We find the iterative screening scheme is fundamentally capable of developing desired aptamers. On the other hand, target sample configuration and separation efficiency may all together significantly diversify the screening dynamics and results.     

Application of a novel in vitro selection technique to isolate and characterise high affinity DNA aptamers binding mammalian prion proteins

Clinical diagnosis and research into transmissible spongiform encephalopathies are hampered by the lack of sufficiently sensitive and specific reagents able to adequately detect the normal cellular form of the prion protein, PrP(C), and the pathological isoform, PrP(Sc). In order to provide such reagents, we applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) against a recombinant murine prion protein, to select single-stranded DNA ligands (aptamers) of high affinity. The SELEX protocol and subsequent aptamer characterisation employed protein immobilisation/partitioning using nickel-complexed magnetic particles and a novel SYBR Green-mediated quantitative real-time PCR technique. Following eight rounds of selection, the enriched aptamer pool was cloned and 24 clones sequenced. Seven of these were 'orphan' clones and the remainder were grouped into three separate T-rich families. All but four of the aptamer clones exhibited specific binding to the murine prion protein and the majority also bound to human and ovine prion proteins. Dissociation constants (K(d)) ranged from 18 to 79 nM. Flow cytometry with fluorescein-labelled aptamers confirmed that binding to cells was dependent on the expression of PrP(C). Preliminary studies also indicate that a trivalent aptamer pool is capable of binding the pathological isoform PrP(Sc) following guanidinium denaturation.     

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.     

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.