Parasite-specific aptamers as biosynthetic reagents and potential pharmaceuticals

Aptamers are short, synthetic nucleic acid molecules. They are generated by a Darwinian-type in vitro evolution method known as 'systematic evolution of ligands by exponential enrichment' (SELEX). SELEX represents an experimental platform to identify rare ligands with predetermined functionality from combinatorial nucleic acid libraries. Since its discovery about 20 years ago the method has been instrumental in identifying a large number of aptamers that recognize targets of very different chemistry and molecular complexity. Although aptamers have been converted into sophisticated biomolecular tools for a diverse set of technologies, only a limited number of aptamers have been selected as binding reagents for parasites or parasite-derived molecules. Here the published examples of aptamers that target Leishmania-, Trypanosoma- and Plasmodia-specific molecules are reviewed.     

Advances in SELEX and application of aptamers in the central nervous system

SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is a screening technique that involves the progressive selection of highly specific ligands by repeated rounds of partition and amplification from a large combinatorial nucleic acid library. The products of the selection are called aptamers, which are short single stranded DNA or RNA molecules, binding with high affinity, attributed to their specific three-dimensional shapes, to a large variety of targets, ranging from small molecules to complex mixtures. Various improvement of the original SELEX method described in 1990 have been obtained recently, such as capillary electrophoresis SELEX, Toggle-SELEX, Tailored-SELEX, Photo-SELEX, and others. These new variants greatly shorten time of selection and improve aptamer affinity and specificity. Such aptamers have great potential as detecting and/or diagnostic reagents. Furthermore, some aptamers specifically inhibit biological functions of targeted proteins, and are considered as potent therapeutic lead structures evaluated in preclinical disease models. Recently, one aptamer has been approved by Food and Drug Administration of US for treating age-related macular degeneration. This review presents recent advances in the field of SELEX with special emphasis on applications of aptamers as analytical, diagnostic and therapeutic tools in the central nervous system.     

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。     

Chemically modified nucleic acid aptamers for in vitro selections: evolving evolution

Combinatorial library selections through the systematic evolution of ligands by exponential enrichment (SELEX) technique identify so-called nucleic acid aptamers that bind with high-affinity and specificity to a wide range of selected molecules. However, the modest chemical functionality of nucleic acids poses some limits on their versatility as binders and catalysts, and, furthermore, the sensitivity of pure RNA- and DNA-based aptamers to nucleases restricts their use as therapeutic and diagnostic agents. Here we review synthetic chemistries for modifying nucleotides that have been developed to enhance the affinity of aptamers for targets and to increase their stability in biological fluids. Implementation of in vitro selections with modified nucleotides promises to be an elegant technique for the creation of ligands with novel physical and chemical properties and is anticipated to have a significant impact on biotechnology, diagnostics and drug development. The current molecular designs and applications of modified nucleotides for in vitro selections are reviewed, along with a discussion of future developments expected to further the utility of this approach in both practical and theoretical terms.     

Trends in the Design and Development of Specific Aptamers Against Peptides and Proteins

Aptamers are single stranded oligonucleotides, comparable to monoclonal antibodies (mAbs) in selectivity and affinity and have significant strategic properties in design, development and applications more than mAbs. Ease of design and development, simple chemical modification and the attachment of functional groups, easily handling and more adaptability with analytical methods, small size and adaptation with nanostructures are the valuable characteristics of aptamers in comparison to large protein based ligands. Among a broad range of targets that their specific aptamers developed, proteins and peptides have significant position according to the number of related studies performed so far. Since proteins control many of important physiological and pathological incidents in the living organisms, particularly human beings and because of the benefits of aptamers in clinical and analytical applications, aptamer related technologies in the field of proteins and peptides are under progress, exclusively. Currently, there is only one FDA approved therapeutic aptamer in the pharmaceutical market, which is specific to vascular endothelial growth factor and is prescribed for age related macular degenerative disease. Additionally, there are several aptamers in the different phases of clinical trials. Almost all of these aptamers are specific to clinically important peptide or protein targets. In addition, the application of protein specific aptamers in the design and development of targeted drug delivery systems and diagnostic biosensors is another intersting field of aptamer technology. In this review, significant efforts related to development and applications of aptamer technologies in proteins and peptides sciences were considered to emphasis on the importance of aptamers in medicinal and clinical applications.     

SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands

SELEX stands for systematic evolution of ligands by exponential enrichment. This method, described primarily in 1990 [Ellington, A.D., Szostak, J.W., 1990. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818-822; Tuerk, C., Gold, L., 1990. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505-510] aims at the development of aptamers, which are oligonucleotides (RNA or ssDNA) binding to their target with high selectivity and sensitivity because of their three-dimensional shape. Aptamers are all new ligands with a high affinity for considerably differing molecules ranging from large targets as proteins over peptides, complex molecules to drugs and organic small molecules or even metal ions. Aptamers are widely used, including medical and pharmaceutical basic research, drug development, diagnosis, and therapy. Analytical and separation tools bearing aptamers as molecular recognition and binding elements are another big field of application. Moreover, aptamers are used for the investigation of binding phenomena in proteomics. The SELEX method was modified over the years in different ways to become more efficient and less time consuming, to reach higher affinities of the aptamers selected and for automation of the process. This review is focused on the development of aptamers by use of SELEX and gives an overview about technologies, advantages, limitations, and applications of aptamers.     

In vitro selection of high-affinity nucleic acid ligands to parasite target molecules

The logic of using nucleic acids as pharmaceutical reagents is in part based on their capacity to interact with high affinity and specificity with other biological components. Considerable progress has been made over the past 10 years in the development of nucleic acid-based drug molecules using a variety of different technologies. One approach is a combinatorial technology that involves an iterative Darwinian-type in vitro evolution process, which has been termed SELEX for 'systematic evolution of ligands by exponential enrichment'. The procedure is a highly efficient method of identifying rare ligands from combinatorial nucleic acid libraries of very high complexity. It allows the selection of nucleic acid molecules with desired functions and it has been instrumental in the identification of a number of synthetic DNA and RNA molecules, so-called aptamers that recognise ligands of different chemical origin. The method is fast, it does not require special equipment and the selected aptamers typically bind their target with high affinity and high specificity. Here we summarise the recent examples of the SELEX technique within the context of identifying high-affinity ligands against parasite target molecules.     

SELEX技术在神经系统疾病研究中的应用

配体指数级富集系统进化（systematic evolution of ligands by exponential enrichment,SELEX）技术是一种组合化学技术,可经过反复筛选扩增得到针对靶分子的高亲和力和高特异性的适配子.适配子通过识别、结合特定靶分子并对其进行功能调控从而达到对疾病诊断和治疗的目的 .近年来SELEX技术在神经系统功能和疾病研究中的应用越来越多.现已经筛选出针对朊蛋白、肌腱蛋白-C、β-淀粉样肽、乙酰胆碱受体的自身抗体等靶标的适配子,促进了对朊病毒病、脑肿瘤、阿茨海默病、重症肌无力等神经系统疾病的诊断和治疗研究,为这些疾病的诊治提供了新的研究工具.     

Differential SELEX in Human Glioma Cell Lines

The hope of success of therapeutic interventions largely relies on the possibility to distinguish between even close tumor types with high accuracy. Indeed, in the last ten years a major challenge to predict the responsiveness to a given therapeutic plan has been the identification of tumor specific signatures, with the aim to reduce the frequency of unwanted side effects on oncologic patients not responding to therapy. Here, we developed an in vitro evolution-based approach, named differential whole cell SELEX, to generate a panel of high affinity nucleic acid ligands for cell surface epitopes. The ligands, named aptamers, were obtained through the iterative evolution of a random pool of sequences using as target human U87MG glioma cells. The selection was designed so as to distinguish U87MG from the less malignant cell line T98G. We isolated molecules that generate unique binding patterns sufficient to unequivocally identify any of the tested human glioma cell lines analyzed and to distinguish high from low or non-tumorigenic cell lines. Five of such aptamers act as inhibitors of specific intracellular pathways thus indicating that the putative target might be important surface signaling molecules. Differential whole cell SELEX reveals an exciting strategy widely applicable to cancer cells that permits generation of highly specific ligands for cancer biomarkers.     

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。     

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.     

Aptamers as reagents for high-throughput screening

The identification of new drug candidates from chemical libraries is a major component of discovery research in many pharmaceutical companies. Given the large size of many conventional and combinatorial libraries and the rapid increase in the number of possible therapeutic targets, the speed with which efficient high-throughput screening (HTS) assays can be developed can be a rate-limiting step in the discovery process. We show here that aptamers, nucleic acids that bind other molecules with high affinity, can be used as versatile reagents in competition binding HTS assays to identify and optimize small-molecule ligands to protein targets. To illustrate this application, we have used labeled aptamers to platelet-derived growth factor B-chain and wheat germ agglutinin to screen two sets of potential small-molecule ligands. In both cases, binding affinities of all ligands tested (small molecules and aptamers) were strongly correlated with their inhibitory potencies in functional assays. The major advantages of using aptamers in HTS assays are speed of aptamer identification, high affinity of aptamers for protein targets, relatively large aptamer-protein interaction surfaces, and compatibility with various labeling/detection strategies. Aptamers may be particularly useful in HTS assays with protein targets that have no known binding partners such as orphan receptors. Since aptamers that bind to proteins are often specific and potent antagonists of protein function, the use of aptamers for target validation can be coupled with their subsequent use in HTS.     

适配体与病毒性感染诊断及治疗

适配体（Aptamers）是通过指数富集的配体系统进化（systematic evolution of ligands by exponential enrichment,SELEX）技术,从随机核酸文库中筛选出来的单链寡核苷酸,已在临床医疗及其他领域得到日益广泛的应用.与抗体相比,适配体具有很多优点,如高亲和力、高特异性、分子量小、几乎无免疫排斥反应、结构稳定、易于合成等.可用于适配体筛选的靶标范围非常广,包括有机小分子、蛋白、完整细胞及病毒颗粒等.迅速可靠的病原检测对于病毒性传染病的成功预防和治疗具有重要意义.随着严格筛选和快速分离技术的进步,适配体在病毒感染的检测治疗中显示出巨大的潜力.本文概括介绍了适配体在病毒研究方面的最新应用进展及未来前景.     

Isolation of specific and high-affinity RNA aptamers against NS3 helicase domain of hepatitis C virus

Hepatitis C virus (HCV)-encoded nonstructural protein 3 (NS3) possesses protease, NTPase, and helicase activities, which are considered essential for viral proliferation. Thus, HCV NS3 is a good putative therapeutic target protein for the development of anti-HCV agents. In this study, we isolated specific RNA aptamers to the helicase domain of HCV NS3 from a combinatorial RNA library with 40-nucleotide random sequences using in vitro selection techniques. The isolated RNAs were observed to very avidly bind the HCV helicase with an apparent Kd of 990 pM in contrast to original pool RNAs with a Kd of >1 microM. These RNA ligands appear to impede binding of substrate RNA to the HCV helicase and can act as potent decoys to competitively inhibit helicase activity with high efficiency compared with poly(U) or tRNA. The minimal binding domain of the ligands was determined to evaluate the structural features of the isolated RNA molecules. Interestingly, part of binding motif of the RNA aptamers consists of similar secondary structure to the 3'-end of HCV negative-strand RNA. Moreover, intracellular NS3 protein can be specifically detected in situ with the RNA aptamers, indicating that the selected RNAs are very specific to the HCV NS3 helicase. Furthermore, the RNA aptamers partially inhibited RNA synthesis of HCV subgenomic replicon in Huh-7 hepatoma cell lines. These results suggest that the RNA aptamers selected in vitro could be useful not only as therapeutic and diagnostic agents of HCV infection but also as a powerful tool for the study of HCV helicase mechanism.     

综述与专论: 基于核酸适配体的细胞外囊泡分离分析方法

细胞外囊泡通过参与细胞间通讯，在诸多生理病理过程中发挥着重要作用。因此，细胞外囊泡的分离分析对理解其生物学功能以及发展基于囊泡的疾病诊疗方法具有重要价值。细胞外囊泡的高效分离以及高灵敏可靠检测很大程度上取决于识别配体。核酸适配体是一类高效、特异结合其靶标分子的单链寡核苷酸。核酸适配体的易修饰和可程序化设计等特征，使其成为细胞外囊泡分离和分析的理想识别配体。为提高细胞外囊泡的分离效率，研究者们提出多种策略用于提升核酸适配体的亲和力，以及界面与细胞外囊泡的接触几率。此外，分离不同亚型的细胞外囊泡有助于理解细胞外囊泡的生物学意义。在细胞外囊泡分析方面，根据核酸适配体与细胞外囊泡识别信号的转导方式不同，分为电化学、可视化、表面增强拉曼光谱、荧光法等方法。本文综述了核酸适配体的筛选以及其在细胞外囊泡分离和分析中的最新进展、挑战及未来方向。     

Generation of inhibitory DNA aptamers against human hepatocyte growth factor

Hepatocyte growth factor (HGF), a multifunctional cytokine, can act on many cell types. It is involved in cancer growth and metastasis by enhancing the motility of cancer cells and stimulating angiogenesis. The development of effective inhibitors for HGF is an important issue in cancer therapy. In this study, we isolated DNA aptamers against human HGF using the systematic evolution of ligands by exponential enrichment method. The selected DNA aptamers had a highly conserved consensus sequence, and could be divided into two major classes (classes I and II). The consensus motif of classes I and II might contribute to the formation of a hairpin loop structure and a G-quartet structure, respectively. These DNA aptamers bound to human HGF with high affinity and specificity. The dissociation constants of typical aptamers H38-15 and H38-21, representative of the two classes, were calculated to be approximately 20 nM. H38-15 and H38-21 inhibited the biological activities of HGF including the stimulation of scattering, migration, and invasion of pancreatic cancer KP-3 cells. Furthermore, both aptamers inhibited HGF-induced tube formation by human umbilical vein endothelial cells. These results suggested that the isolated DNA aptamers will be useful as therapeutic and diagnostic reagents for cancers.     

RNA and DNA aptamers in cytomics analysis.

BACKGROUND: The systematic evolution of ligands by exponential enrichment (SELEX) technique is a combinatorial library approach in which DNA or RNA molecules (aptamers) are selected by their ability to bind their protein targets with high affinity and specificity, comparable to that of monoclonal antibodies. In contrast to antibodies conventionally selected in animals, aptamers are generated by an in vitro selection process, and can be directed against almost every target, including antigens like toxins or nonimmunogenic targets, against which conventional antibodies cannot be raised. METHODS: Aptamers are ideal candidates for cytomics, as they can be attached to fluorescent reporters or nanoparticles in order to study biological function by fluorescence microscopy, by flow cytometry, or to quantify the concentration of their target in biological fluids or cells using ELISA, RIA, and Western blot assays. RESULTS: We demonstrate the in vitro selection of anti-kinin B1 receptor aptamers that could be used to determine B1 receptor expression during inflammation processes. These aptamers specifically recognize their target in a Northern-Western blot assay, and bind to their target protein whenever they are exposed in the membrane. CONCLUSIONS: Currently, aptamers are linked to fluorescent reporters. We discuss here the present status and future directions concerning the use of the SELEX technique in cytomics.     

Nucleic acid aptamers for target validation and therapeutic applications.

In the simplest view, aptamers can be thought of as nucleic acid analogs to antibodies. They are able to bind specifically to proteins, and, in many cases, that binding leads to a modulation of protein activity. New aptamers are rapidly generated through the SELEX (Systematic Evolution of Ligands by Exponential enrichment) process and have a very high target affinity and specificity (picomoles to nanomoles). Furthermore, aptamers composed of modified nucleotides have a long in vivo half-life (hours to days), are nontoxic and nonimmunogenic, and are easily produced using standard nucleic acid synthesis methods. These properties make aptamers ideal for target validation and as a new class of therapeutics. As a target validation tool, aptamers provide important information that complements that provided by other methods. For example, siRNA is widely used to demonstrate that protein knock-out in a cellular assay can lead to a biological effect. Aptamers extend that information by showing that the dose-dependent modulation of protein activity can be used to derive a therapeutic benefit. That is, aptamers can be used to demonstrate that the protein is a good target for drug development. As a new class of therapeutics, aptamers bridge the gap between small molecules and biologics. Like biologics, biologically active aptamers are rapidly discovered, have no class-specific toxicity, and are adept at disrupting protein-protein interaction. Like small molecules, aptamers can be rationally engineered and optimized, are nonimmunogenic, and are produced by scalable chemical procedures at moderate cost. As such, aptamers are emerging as an important source of new therapeutic molecules.     

Expanding the application potential of DNA aptamers by their functionalization

Side-by-side development of two competing technologies for obtaining affinity antibody-based and aptamer-based molecules opens new horizons for the creation of diagnostic and therapeutic agents of extremely high efficiency. Benefits of aptamers, such as relatively small size and selection simplicity, have been jeopardized for a long time by their intrinsic downsides, i.e., obscure process of obtaining aptamers against certain targets because of a low diversity of functional groups (purine and pyrimidine bases) in DNA and RNA aptamers. Another side effect of the aptamer technique inherent to the traditional SELEX method is unspecific enrichment with aptamers with high affinity to off-target reaction components. Today, due to current progress in the development of new technology methods and chemical coupling reactions, the modern aptamer technology helps to avoid its disadvantages and become capable of being the source of new diagnostic and therapeutic tools, which are properly unique in their efficiency. The review focuses on modern methods of increasing efficiency of the aptamer selection and on synthetic nucleotide modifications, which make it possible to prepare high-affinity aptamers against traditionally ‘hard’ targets.     

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 (Fe(3)O(4)) 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.