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.     

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.     

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 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 against extracellular targets for in vivo applications

Oligonucleotides are multifunctional molecules which can interfere with gene expression by different mechanism such as antisense, RNA interference, ribozymes, etc. For most in vivo diagnostic and therapeutic applications, oligonucleotides need to be delivered to the intracellular compartment of a specific organ, a difficult task which limits considerably their use. However, aptamer oligonucleotides which target extracellular markers obviate this problem. Aptamers are short oligonucleotides (<100 bases) selected from large combinatorial pools of sequences for their capacity to bind to many types of 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 on their targets. Moreover, they seem to lack immunogenicity and can be chemically modified in order to improve their stability against nucleases or extend their blood circulation time, two properties which are particularly useful for in vivo applications. Recently, aptamers have been selected against whole living cells, opening a new avenue which presents three major advantages 1) direct selection without prior purification of the targets; 2) conservation of membrane proteins in their native conformation similar to the in vivo conditions and 3) identification of (new) targets for a specific phenotype. Many aptamers are now being developed against biomedical relevant extracellular targets: membrane receptor proteins, hormones, neuropeptides, coagulation factors... Among them, one aptamer that inhibits the human VEGF165 has recently been approved by FDA for the treatment of age-related macular degeneration. Here we discuss the recent developments of aptamers against extracellular targets for in vivo therapy and as tools for diagnosis using molecular imaging.     

Conservative secondary structure motif of streptavidin-binding aptamers generated by different laboratories

Aptamers that are selected in vitro from random pools of DNA or RNA molecules by SELEX (Systematic evolution of ligands by exponential enrichment) technique have been extensively explored for analytical and biomedical applications. Although many aptamers with high affinity and specificity against specific ligands have been reported, there is still a lack of well characterized DNA aptamers. Here we report the selection of a group of aptamer candidates (85 mer) against streptavidin. Through comparing the predicted secondary structures of all the candidates, a conservative bulge-hairpin structure section (about 29 mer) was found, and then it was determined to be the binding motif to streptavidin. This binding motif was further discovered to also exist in streptavidin-binding aptamers (SBAs) selected by three other laboratories using different methods. The primary sequences of this secondary structure motif are very different, only several nucleotides in the loop and bulge area are critical for binding and other nucleotides are variable. The streptavidin binding of all the SBAs could be competed by biotin implying that they bind to the same site on streptavidin. These results suggest that the evolution of SBA is predominated by specific groups on streptavidin. The highly variable sequence composition of streptavidin-binding aptamer would make the design of aptameric sensor or device based on streptavidin more flexible and easy.     

Methods for selection of aptamers to protein targets

Aptamers are synthetic single-stranded RNA or DNA molecules capable of specific binding to other target molecules. In this review, the main aptamer properties are considered and methods for selection of aptamers against various protein targets are described. Special attention is given to the methods for directed selection of aptamers, which allow one to obtain ligands with specified properties.     

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.     

In vivo uses of aptamers selected against cell surface biomarkers for therapy and molecular imaging

Nucleic acid Aptamers are ligands that are selected by a process of molecular evolution to bind with high affinities and specificities to a specific target. Recently, an increasing number of aptamers have been selected against biomarkers expressed at the surface of human cells or infectious pathogens. This class of targets, mostly proteins, is associated with several pathologies including cancer, inflammation and infection diseases. Several of these cell surface specific aptamers were tested in vivo as drugs or as targeting agents for nanocarriers, siRNA or contrast agents. Strikingly, they were used to develop a wide variety of new treatments or new approaches for molecular imaging and they were also able to improve current therapies such as chemotherapy, radiotherapy or immunotherapy. This review presents these different applications and the different studies conducted in vivo with this class of aptamers, predominantly in pre-clinical models.     

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.     

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.     

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.     

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.     

DNA-aptamer/protein interaction as a cause of apoptosis and arrest of proliferation in Ehrlich ascites adenocarcinoma cells

New prospects for the applications of single-stranded DNA and RNA as therapeutic agents have been discovered in the recent years. Aptamers are the oligonucleotides that bind to their targets with high affinity and specificity due to the well-defined tertiary structures and spatial charge distribution. Aptamers can be selected for any molecules, virus particles, bacteria, cells, and tissues. They have a wide range of applications from target identification to drug delivery. Aptamers themselves can affect various cell functions by affecting certain proteins and receptors. Here, we present the technique for selecting aptamers with antitumor activity in cancer cell cultures and identifying their target proteins by mass spectrometry analysis. The evolved aptamers showed the following antitumor properties: AS-14 (K _d = 3.8 nM) induced apoptosis (phosphatidylserine translocation determined with Annexin V Alexa Fluor 488) and AS-9 (K _d = 0.75 nM) stopped proliferation (as determined with CellTrace? Far Red DDAO-SE) in the culture of Ehrlich ascites adenocarcinoma cells. Using high performance liquid chromatography and high resolution tandem mass spectrometry, we have identified the proteins affected by the AS-14 and AS-9 aptamers. One of the most likely targets for AS-14 was filamin A, which is involved in metastasis formation, tumor development, and cell proliferation. According to mass spectrometry data, the AS-9 aptamer influences the α-subunit of mitochondrial ATP synthase, the key component of mitochondrial oxidative phosphorylation, stimulation of which leads to tumor growth suppression. Thus, mass spectrometry data confirmed the results of the experiments on cell cultures showing that the aptamer binding to specific protein targets causes apoptosis and stops proliferation of cancer cells. However, the mechanisms of action of aptamers in vitro and in vivo are not clear enough and still need to be determined. Our study opens up new possibilities for creation of non-toxic drugs based on DNA aptamers for targeted anticancer therapy.     

Aptamers: versatile probes for flow cytometry

Aptamers are nucleic acid oligomers with distinct conformational shapes that allow them to bind targets with high affinity and specificity. Aptamers are selected from a random oligonucleotide library by their capability to bind a certain molecular target. A variety of targets ranging from small molecules like amino acids to complex targets and whole cells have been used to select aptamers. These characteristics and the ability to create specific aptamers against virtually any cell type in a process termed “systematic evolution by exponential enrichment” make them interesting tools for flow cytometry. In this contribution, we review the application of aptamers as probes for flow cytometry, especially cell-phenotyping and detection of various cancer cell lines and virus-infected cells and pathogens. We also discuss the potential of aptamers combined with nanoparticles such as quantum dots for the generation of new multivalent detector molecules with enhanced affinity and sensitivity. With regard to recent advancements in aptamer selection and the decreasing costs for oligonucleotide synthesis, aptamers may rise as potent competitors for antibodies as molecular probes in flow cytometry.     

Nanotechnology and aptamers: applications in drug delivery

Nucleic acid ligands, also known as aptamers, are a class of macromolecules that are being used in several novel nanobiomedical applications. Aptamers are characterized by high affinity and specificity for their target, a versatile selection process, ease of chemical synthesis and a small physical size, which collectively make them attractive molecules for targeting diseases or as therapeutics. These properties will enable aptamers to facilitate innovative new nanotechnologies with applications in medicine. In this review, we will highlight recent developments in using aptamers in nanotechnology solutions for treating and diagnosing disease.     

Analytical applications of aptamers

So far, several bio-analytical methods have used nucleic acid probes to detect specific sequences in RNA or DNA targets through hybridisation. More recently, specific nucleic acids, aptamers, selected from random sequence pools, have been shown to bind non-nucleic acid targets, such as small molecules or proteins. The development of in vitro selection and amplification techniques has allowed the identification of specific aptamers, which bind to the target molecules with high affinity. Many small organic molecules with molecular weights from 100 to 10,000 Da have been shown to be good targets for selection. Moreover, aptamers can be selected against difficult target haptens, such as toxins or prions. The selected aptamers can bind to their targets with high affinity and even discriminate between closely related targets.

Aptamers can thus be considered as a valid alternative to antibodies or other bio-mimetic receptors, for the development of biosensors and other analytical methods. The production of aptamers is commonly performed by the SELEX (systematic evolution of ligands by exponential enrichment) process, which, starting from large libraries of oligonucleotides, allows the isolation of large amounts of functional nucleic acids by an iterative process of in vitro selection and subsequent amplification through polymerase chain reaction.

Aptamers are suitable for applications based on molecular recognition as analytical, diagnostic and therapeutic tools. In this review, the main analytical methods, which have been developed using aptamers, will be discussed together with an overview on the aptamer selection process.     

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。     

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.