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。     

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。     

适配体结合蛋白质靶标的亲和力表征方法及其在疾病诊断中的应用

核酸适配体是通过体外指数富集配体系统进化（SELEX）技术筛选获得,并能够和蛋白质靶标高特异性、高亲和力结合的单链寡核苷酸。核酸适配体不但具有抗体的识别特性,而且具有自己独特的优良性能,目前已应用于分析检验、食品安全和生物医药等各个领域。蛋白质具有多种多样的生物功能以及临床诊断价值。因此,核酸适配体针对蛋白质靶标并在蛋白质相关的基础研究领域受到广泛的关注。核酸适配体应用性能的优劣取决于与其靶标蛋白质的亲和力与特异性。本文主要综述核酸适配体对蛋白质靶标的亲和力表征方法,以及在药物研发、肿瘤检测、生物成像以及生物传感器方面的应用。     

DNA适配体与金属离子的结合机理的探究

核酸适配体是一类经过体外筛选获得的功能性寡核苷酸序列片段,能与特定的靶标进行高特异性与强亲和力的结合,以金属离子作为靶标的核酸适配体是适配体技术发展的一个重要研究方向。本文综述了近些年来国内外核酸适配体与金属离子的结合机理,并对该领域目前存在的问题进行分析和展望。     

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。     

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。     

核酸适配体在肿瘤诊断和治疗中的应用研究

随着科学技术的飞速发展,适配体在各个领域的研究也备受关注,特别是在肿瘤治疗研究方面。核酸适配体是一类通过指数富集的配体系统进化技术(systematic evolution of ligands by exponential enrichment technology,SELEX)获得的,具有独特三维构象的单链DNA或小分子RNA。核酸适配体能高亲和力和高特异性的与靶点结合,同时具有无免疫原性、相对分子质量小、靶标分子范围广、热学及化学稳定性高等特点。目前核酸适配体广泛应用于疾病的诊断、治疗、生物标志物筛选、生物传感器、新药研发等领域。现将核酸适配体的特点、筛选、及在肿瘤诊断和治疗中的研究作一综述。     

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。     

小分子靶标与其核酸适配体亲和力的表征方法

核酸适配体是指通过SELEX筛选得到的能与靶标高特异性、高亲和力结合的单链DNA或RNA。目前国内外具有高亲和性和高特异性结合的小分子靶标的核酸适配体依然很少,究其原因,一方面是因为小分子靶标的适配体难以筛选,另一方面是小分子靶标与其候选适配体亲和力表征方法难以确定。亲和力表征是确定适配体筛选成功与否的关键步骤,就现有的小分子靶标与其相应适配体亲和力表征方法进行了总结,包括纳米金比色法、等温滴定量热法、表面等离子共振、圆二色谱法、石英晶体微天平法、微量热泳动法和SYBR Green I染料检测法等,并分析了这些方法的优缺点及改进建议,以期有助于提高适配体表征效率。     

核酸适配体筛选技术研究进展

核酸适配体是一段特殊的寡核苷酸配基,对靶标有亲和力和特异性。核酸适配体发展十分迅速,但筛选方法的研究相对来说还不能满足适配体应用的需求。笔者从筛选过程出发,针对近年来较为新颖热门的非天然核苷酸替代传统核苷酸、镜像核酸库代替核酸库等核酸库优化方法,毛细管电泳筛选、微流控芯片技术、微阵列芯片、粒子展示等筛选策略改进技术,高通量测序等次级文库序列分析,碱基突变与修饰、序列截短等适配体结构优化等具体内容进行总结,探讨各种方法的研究进展及应用前景。     

肝脏去唾液酸糖蛋白受体特异性RNA适配子的SELEX筛选与鉴定

目的获得能够特异性高亲和力结合肝脏特异性去唾液酸糖蛋白受体（asialoglycoprotein receptor,ASGPR）的RNA适配子,为开发诊断和治疗肝脏疾病的靶向性试剂和药物奠定基础。方法合成一个长度为115nt含有25个随机序列的单链DNA随机文库,通过体外转录构建出单链RNA适配子随机文库,以肝脏ASGPR大亚基为靶蛋白,采用SELEX（systematic evolution of ligands by exponential enrichment）技术筛选具有高亲和力的AsGPR特异性RNA适配子;通过膜结合测定实验、凝胶阻滞实验鉴定筛选适配子对靶蛋白的特异性和亲和力。结果经过12轮筛选获得了具有高亲和力的肝脏ASGPR特异性RNA适配子。结论成功地筛选出了具有离亲和力的肝脏ASGPR特异性RNA适配子库。     

Screening and Characterization of a Novel RNA Aptamer That Specifically Binds to Human Prostatic Acid Phosphatase and Human Prostate Cancer Cells

Prostatic acid phosphatase (PAP) expression increases proportionally with prostate cancer progression, making it useful in prognosticating intermediate to high-risk prostate cancers. A novel ligand that can specifically bind to PAP would be very helpful for guiding prostate cancer therapy. RNA aptamers bind to target molecules with high specificity and have key advantages such as low immunogenicity and easy synthesis. Here, human PAP-specific aptamers were screened from a 2′-fluoropyrimidine (FY)-modified RNA library by SELEX. The candidate aptamer families were identified within six rounds followed by analysis of their sequences and PAP-specific binding. A gel shift assay was used to identify PAP binding aptamers and the 6N aptamer specifically bound to PAP with a Kd value of 118 nM. RT-PCR and fluorescence labeling analyses revealed that the 6N aptamer bound to PAP-positive mammalian cells, such as PC-3 and LNCaP. IMR-90 negative control cells did not bind the 6N aptamer. Systematic minimization analyses revealed that 50 nucleotide sequences and their two hairpin structures in the 6N 2′-FY RNA aptamer were equally important for PAP binding. Renewed interest in PAP combined with the versatility of RNA aptamers, including conjugation of anti-cancer drugs and nano-imaging probes, could open up a new route for early theragnosis of prostate cancer.     

Structural optimization of an aptamer generated from Ligand-Guided Selection (LIGS) resulted in high affinity variant toward mIgM expressed on Burkitt's lymphoma cell lines

Aptamers are synthetic, short nucleic acid molecules capable of specific target recognition. Aptamers are selected using a screening method termed Systematic Evolution of Ligands by Exponential enrichment (SELEX). We recently have introduced a variant of SELEX called “Ligand-Guided-Selection” (LIGS) that allows the identification of specific aptamers against known cell-surface proteins. Utilizing LIGS, we introduced three specific aptamers against membrane-bound IgM (mIgM), which is the hallmark of B cells. Out of the three aptamers selected against mIgM, an aptamer termed R1, in particular, was found to be interesting due to its ability to recognize mIgM on target cells and then block anti-IgM antibodies binding their antigen. We systematically truncated parent aptamer R1 to design shorter variants with enhanced affinity. Importantly, herein we show that the specificity of the most optimized variant of R1 aptamer is similar to that of anti-IgM antibody, indicating that the specificity of the ligand utilized in selective elution of the aptamer determines the specificity of the LIGS-generated aptamer. Furthermore, we report that truncated variants of R1 are able to recognize mIgM-positive human B lymphoma BJAB cells at physiological temperature, demonstrating that LIGS-generated aptamers could be re-optimized into higher affinity variants. Collectively, these findings show the significance of LIGS in generating highly specific aptamers with potential applications in biomedicine.     

A cell-based single-stranded DNA aptamer specifically targets gastric cancer

Gastric cancer is one of the most prevailing cancers with high morbidity and mortality. Limitations in the current diagnosis and therapy, specially lacking of specific molecular therapeutic targets, ask for the development of new strategies. Aptamer, a newly developed adaptive molecule, could be used in clinical detection and therapy because of its high affinity and specificity. As no aptamer has ever been developed in preventing gastric cancer so far, we were the first who cloned such an aptamer specifically targeting gastric cancer. The aptamer was selected by systematic evolution of ligands by exponential enrichment with gastric cancer cell-line HGC-27 as target cell line and immortalized gastric epithelial cell-line GES-1 as control cell line. The affinity and specificity of candidate aptamers were examined by flow cytometry, confocal imagining and aptamer-based histochemistry staining. After 19 cycles of systematic evolution of ligands by exponential enrichment and subsequent cloning and sequencing, an aptamer with the highest affinity and specificity (nominated as AGC03) among candidates was screened out from a random single-stranded DNA pool. Moreover, AGC03 could not only specifically bind to gastric cancer cells (the equilibrium dissociation constant value was 16.49 ± 0.40 nM) in vitro, but also recognize cancer cells in human cancer tissue. Our most important finding is that AGC03 could even be internalized into cells automatically. In conclusion, we obtained a novel aptamer specifically targeting gastric cancer, which is an effective tool for both gastric cancer diagnosis and drug delivery.     

Chimeric aptamers in cancer cell-targeted drug delivery

Aptamers are single-stranded structured oligonucleotides (DNA or RNA) that can bind to a wide range of targets (“apatopes”) with high affinity and specificity. These nucleic acid ligands, generated from pools of random-sequence by an in vitro selection process referred to as systematic evolution of ligands by exponential enrichment (SELEX), have now been identified as excellent tools for chemical biology, therapeutic delivery, diagnosis, research, and monitoring therapy in real-time imaging. Today, aptamers represent an interesting class of modern pharmaceuticals which with their low immunogenic potential mimic extend many of the properties of monoclonal antibodies in diagnostics, research, and therapeutics. More recently, chimeric aptamer approach employing many different possible types of chimerization strategies has generated more stable and efficient chimeric aptamers with aptamer–aptamer, aptamer–nonaptamer biomacromolecules (siRNAs, proteins) and aptamer–nanoparticle chimeras. These chimeric aptamers when conjugated with various biomacromolecules like locked nucleic acid (LNA) to potentiate their stability, biodistribution, and targeting efficiency, have facilitated the accurate targeting in preclinical trials. We developed LNA-aptamer (anti-nucleolin and EpCAM) complexes which were loaded in iron-saturated bovine lactofeerin (Fe-blf)-coated dopamine modified surface of superparamagnetic iron oxide (Fe₃O₄) nanoparticles (SPIONs). This complex was used to deliver the specific aptamers in tumor cells in a co-culture model of normal and cancer cells. This review focuses on the chimeric aptamers, currently in development that are likely to find future practical applications in concert with other therapeutic molecules and modalities.     

Crystal structure of an RNA aptamer bound to thrombin

Aptamers, an emerging class of therapeutics, are DNA or RNA molecules that are selected to bind molecular targets that range from small organic compounds to large proteins. All of the determined structures of aptamers in complex with small molecule targets show that aptamers cage such ligands. In structures of aptamers in complex with proteins that naturally bind nucleic acid, the aptamers occupy the nucleic acid binding site and often mimic the natural interactions. Here we present a crystal structure of an RNA aptamer bound to human thrombin, a protein that does not naturally bind nucleic acid, at 1.9 A resolution. The aptamer, which adheres to thrombin at the binding site for heparin, presents an extended molecular surface that is complementary to the protein. Protein recognition involves the stacking of single-stranded adenine bases at the core of the tertiary fold with arginine side chains. These results exemplify how RNA aptamers can fold into intricate conformations that allow them to interact closely with extended surfaces on non-RNA binding proteins.     

DNA aptamers that bind to PrP(C) and not PrP(Sc) show sequence and structure specificity

DNA aptamers were selected against recombinant human (rhu) cellular prion protein (PrP(C)) 23-231 by systematic evolution of ligands via a systematic evolution of ligands by exponential (SELEX) enrichment procedure using lateral flow chromatography. The SELEX procedure was performed with an aptamer library consisting of a randomized 40-nucleotide core flanked by 28-mer primer-binding sites that, theoretically, represented approximately 10(24) distinct nucleic acid species. Sixty nanograms of rhuPrP(C)23-231 immobilized in the center of a lateral flow device was used as the target molecule for SELEX. At the end of 6 iterations of SELEX, 13 distinct candidate aptamers were identified, of which, 3 aptamers represented 32%, 8%, and 5% of the sequences respectively. Eight aptamers, including the three most frequently occurring candidates, were selected for further evaluation. Selected aptamers bound to rhuPrP(C)23-231 at 10(-6) M to 10(-8) M concentrations. Two of the eight aptamers bound at higher concentrations to rhuPrP(C)90-231. Theoretical thermodynamic modeling of selected aptamer sequences identified several common motifs among the selected aptamers that could play a role in PrP binding. Binding affinity to rhuPrP(C)23-231 was both aptamer sequence and structure dependent. Further, selected aptamers bound to mammalian PrPs derived from brain of healthy sheep, calf, piglet, and deer, and to PrP(C) expressed in mouse neuroblastoma cells. None of the aptamers bound to proteinase K-digested scrapie-infected mouse neuroblastoma cells or untreated PrP-null cells, which further confirmed the PrP(C) specificity of the aptamers. In summary, we enriched and selected DNA aptamers that bind specifically to rhuPrP(C) and mammalian PrP(C) with varying affinities and can be applied to biological samples for PrP(C) enrichment and as diagnostic tools in double ligand assay systems.