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。     

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。     

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。     

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。     

核酸适配体技术及其在肿瘤诊断和治疗中的应用

核酸适配体是一类通过指数富集的配体系统进化（SELEX）技术获得的具有独特三维构象的小分子RNA 或单链DNA。核酸适 配体能高亲和力和高特异性与靶点结合,同时具有自身分子质量小、免疫原性低、热/化学稳定性高、靶标分子范围广等特点,广泛应用 于疾病诊断、治疗、生物传感器、生物标志物筛选、新药研发等领域。综述近年来核酸适配体在肿瘤诊断和治疗方面的应用,并对核酸 适配体的临床研究现状、市场前景及面临挑战和发展趋势作简要分析。     

适配体在受体和分子传感器中的应用研究进展

适配体是从随机寡核苷酸文库中筛选得到的一类可以结合特异靶分子的寡核苷酸序列,在生物医学中具有极其诱人的应用前景.与传统抗体相比,由于具有制备简便、易于修饰、稳定性好,以及结合目标物范围广等特点,适配体已广泛应用于生物传感器的开发,一方面可以用作敏感元件,用于实现对靶分子的识别和检测;另一方面,可以作为敏感元件与传感器件耦合的桥梁,提高耦合的效率.此外,近年来在基于适配体的生物传感器中引入各种纳米材料例如金属纳米颗粒、碳材料和功能性纳米球等也促进了基于适配体的生物传感器在分析性能和商业应用等方面的提升和改进.本文首先简要介绍了基于SELEX技术筛选适配体的方法,总结了适配体的基本特性;其次,详细概括和总结了适配体生物传感器的基本原理及其分类;随后,结合近年来基于适配体的生物传感器领域的最新研究成果,重点介绍了适配体在生物传感器开发中的应用,主要包括在仿生嗅觉与味觉受体传感器方面的应用以及在检测小分子物质和蛋白质的应用;最后,对适配体在生物传感器中的应用面临的问题及其发展趋势进行了分析和展望.     

综述与专论: 基于核酸适配体传感器的即时检测（POCT）技术

即时检测（point-of-care testing，POCT）是一种检测成本低、检测速度快、准确度高、能自我采样获得临床诊断结果的新型诊断技术。该技术在临床诊断、病情监控与疫情防控等领域发挥了重要作用。核酸适配体是一种能够特异性识别多种靶标的分子探针，具有易合成、批间差异小、易实现信号放大等突出优势，是生物医学传感器中重要的分子识别元件。本文概述了核酸适配体探针的现有筛选方法和进展，总结了核酸适配体POCT传感器信号放大策略，着重介绍了各类核酸适配体传感器在POCT领域的应用现状，并对核酸适配体POCT传感器的发展前景进行了展望。     

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。     

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。     

基于核酸适配体的生物分析新方法研究进展

核酸适配体是从随机文库中采用SELEX技术筛选所得的单链短链寡核苷酸片段(通常为15-80个ss DNA或ss RNA)。其能够折叠形成独特稳定的三维结构,通过静电相互作用,氢键,范德华力,碱堆叠或多种作用力组合特异性地与多种靶标结合。适配体因具有构象变化能力而被用作生物分析中的理想识别配体。目前,基于适配体的生物分析新方法得到广泛研究,并用于蛋白多肽类药物分析、疾病标志物诊断、外泌体检测、循环肿瘤细胞检测和病毒检测等方面。本文综述了核酸适配体用于生物分析方法开发的最新进展,比较和讨论不同分析方法,并对基于适配体的生物分析新方法提出了设想和展望,为开发新的生物分析方法和检测技术提供了思路和借鉴。     

Aptamers in clinical diagnostics

Aptamers are single-stranded DNA or RNA oligonucleotides selected in vitro from combinatorial libraries in a process called SELEX (Systematic Evolution of Ligands by EXponential Enrichment). Aptamers play a role of artificial nucleic acid ligands that can recognize and bind to various organic or inorganic target molecules with high specificity and affinity. They can discriminate even between closely related targets and can be easily chemically modified for radioactive, fluorescent and enzymatic labeling or biostability improvement. Aptamers can thus be considered as universal receptors that rival antibodies in diagnostics as a tool of molecular recognition. To date aptamers have been successively used instead of monoclonal antibodies in flow cytometry, immunochemical sandwich assays and in vivo imaging as well to detect wide range of small or large biomolecules.     

Aptamers as functional nucleic acids:<Emphasis Type="Italic">In vitro</Emphasis> selection and biotechnological applications

Aptamers are functional nucleic acids that can specially bind to proteins, peptides, amino acids, nucleotides, drugs, vitamins and other organic and inorganic compounds. The aptamers are identified from random DNA or RNA libraries by a SELEX (systematic evolution of ligands by exponential amplification) process. As aptamers have the advantage, and potential ability to be released from the limitations of antibodies, they are attractive to a wide range of therapeutic and diagnostic applications. Aptamers, with a high-affinity and specificity, could fulfil molecular the recognition needs of various fields in biotechnology. In this work, we reviewed some aptamer selection techniques, properties, medical applications of their molecules and their biotechnological applications, such as ELONA (enzyme linked oligonucleotide assay), flow cytometry, biosensors, electrophoresis, chromatography and microarrays.     

核酸适配体在靶向药物传递中的研究进展

抗癌药物的毒副作用限制了其临床应用,纳米药物载体可实现药物在病灶部位的聚集而不影响正常组织,从而降低药物毒副作用.在药物载体表面修饰靶向配体,以提高药物载体主动靶向进入到细胞的能力,可有效地将药物释放到靶细胞,大大提高药效.核酸适配体(aptamer)作为一种新型的靶向分子,近几年已被运用到靶向药物传递的研究中.本文介绍了几种适配体靶向载药体系,如适配体-药物、适配体-脂质体、适配体-聚合物胶束、适配体-聚合物纳米颗粒、适配体-金属颗粒以及适配体-支化聚合物等载药体系,并对当前研究的热点以及存在的问题和不足进行了评述.     

Aptamers-based assays for diagnostics, environmental and food analysis

Aptamers are single stranded DNA or RNA ligands which can be selected for different targets starting from a huge library of molecules containing randomly created sequences. Aptamers have been selected to bind very different targets, from proteins to small organic dyes. In addition to the very important aspect of having an unlimited source of identical affinity recognition molecules available due to the selection process, aptamers can offer advantages over antibodies that make them very promising for analytical applications. The use of aptamers as therapeutic tools is nowadays well established. On the contrary, the analytical application of aptamers in diagnostic devices or in systems for environmental and food analysis, is still under investigation and the scientific community still need further research to demonstrate the advancements brought by this new kind of ligands. This review will focus on these latter applications with particular attention to the detection of food pathogens, terrorism threat agents, thrombin and cytokines.     

Protein detection via direct enzymatic amplification of short DNA aptamers

Aptamers are single-stranded nucleic acids that fold into defined tertiary structures to bind target molecules with high specificities and affinities. DNA aptamers have garnered much interest as recognition elements for biodetection and diagnostic applications due to their small size, ease of discovery and synthesis, and chemical and thermal stability. Here we describe the design and application of a short DNA molecule capable of both protein target binding and amplifiable bioreadout processes. Because both recognition and readout capabilities are incorporated into a single DNA molecule, tedious conjugation procedures required for protein-DNA hybrids can be omitted. The DNA aptamer is designed to be amplified directly by either polymerase chain reaction (PCR) or rolling circle amplification (RCA) processes, taking advantage of real-time amplification monitoring techniques for target detection. A combination of both RCA and PCR provides a wide protein target dynamic range (1 microM to 10 pM).     

Assays for aptamer-based platforms

Aptamers are single stranded DNA or RNA oligonucleotides that have high affinity and specificity towards a wide range of target molecules. Aptamers have low molecular weight, amenable to chemical modifications and exhibit stability undeterred by repetitive denaturation and renaturation. Owing to these indispensable advantages, aptamers have been implemented as molecular recognition element as alternative to antibodies in various assays for diagnostics. By amalgamating with a number of methods that can provide information on the aptamer-target complex formation, aptamers have become the elemental tool for numerous biosensor developments. In this review, administration of aptamers in applications involving assays of fluorescence, electrochemistry, nano-label and nano-constructs are discussed. Although detection strategies are different for various aptamer-based assays, the core of the design strategies is similar towards reporting the presence of specific target binding to the corresponding aptamers. It is prognosticated that aptamers will find even broader applications with the development of new methods of transducing aptamer target binding.     

Advances in the Application of Modified Nucleotides in SELEX Technology

Aptamers are widely used as molecular recognition elements for detecting and blocking functional biological molecules. Since the common “alphabet” of DNA and RNA consists of only four letters, the chemical diversity of aptamers is less than the diversity of protein recognition elements built of 20 amino acids. Chemical modification of nucleotides enlarges the potential of DNA/RNA aptamers. This review describes the latest achievements in a variety of approaches to aptamers selection with an extended genetic alphabet.     

与细胞因子特异结合的寡聚核苷酸适配子及其在诊断和治疗中的应用

寡聚核苷酸适配子（Aptamer）是用指数富集式配基系统进化方法（SELEX）筛选出的寡聚核苷酸,它能与靶分子特异性结合,具有识别和抑制靶物质生物学活性的作用。将体外筛选到的寡聚核苷酸适配子作为在动物或人体内应用的药剂,还需要进行化学修饰来提高它的生物利用度和在血浆中的稳定性。2氟、2′烷氧基或2′氨基修饰可以提高适配子的稳定性,使适配子的体外半衰期延长;5′端交联一个高分子量的PEG分子或脂质体分子,可以使它的血浆清除率由1小时提高到几小时至1天。修饰后仍保持生物学活性的适配子可用于治疗相应靶细胞因子引起的疾病。目前,国内外已经筛选到了十几种细胞因子的适配子,其中血管内皮生长因子已经用于临床疾病的治疗。除了用于临床治疗外,适配子还可以用于细胞因子的诊断,凡是涉及抗体的诊断领域,几乎都可以用寡聚核苷酸适配子代替。应用大规模机械化筛选技术,可以在短期内筛选到大量的高特异性、高亲和力适配子,这将有力推动临床诊断和治疗的发展。     

139 Development and application of crop exudate specific aptamers

The fertilizer industry is lucrative, though it faces environmental challenges. The amount of nitrogen applied to crops far exceeds the nitrogen utilized by crops leading to excess nitrogen in the form of nitrates, gaseous ammonia, and nitrogen oxides (DeRossa et al., 2010). This excess nitrogen can spread into groundwater contaminating drinking water and causing excess algal growth. Developments in nanotechnology may alleviate some of these environmental challenges. Although there are examples of nanotechnology being utilized for fertilizer products, none of these methods are able to respond to the specific nutrient needs of the plant. This project aims to produce a nanofertilizer product that can synchronize the release of its nutrients with the uptake of nutrients by the plant (DeRossa et al., 2010). Aptamers are synthetic molecules of DNA or RNA that can form 3-D shapes, which are capable of strongly and selectively binding a target of interest. Aptamers have been found to have binding affinities similar to, if not surpassing, those of monoclonal antibodies (Sultan et al., 2009). The goal of this project is to use polyelectrolyte microcapsules containing aptamers in their walls that are specific for key plant signals. This will allow the delivery of nutrient molecules from inside the microcapsules only when required by the plants. Root exudate specific aptamers are being developed using SELEX (Systematic Evolution of Ligands through Exponential enrichment) from a random DNA pool, as well as from an existing aptamer pool. These aptamers will act as molecular recognition probes in the development of an intelligent fertilizer system. Progress from these selections will be presented.     

核酸适配体介导的多药耐药性肿瘤的治疗

化疗是目前肿瘤治疗最常见的方法。然而,肿瘤细胞的多药耐药（multidrug resistance,MDR）常导致临床化疗失败及患者的死亡。因此,干预和逆转肿瘤多药耐药,提高化疗效果,对于肿瘤的治疗具有重要的意义。核酸适配体是一种短的单链寡核苷酸,通过折叠形成特定空间结构从而与靶标特异性结合。靶向肿瘤的核酸适配体可以选择性地将治疗性物质（抗癌药物,siRNA,miRNA）和药物载体递送至肿瘤中,对肿瘤进行靶向杀伤。利用核酸适配体靶向多药耐药性肿瘤,能够特异性干预甚至逆转肿瘤的多药耐药性。本文概述了核酸适配体介导的干预与逆转肿瘤多药耐药性的研究进展。