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.     

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。     

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.     

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.     

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.     

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.     

The selection of aptamers specific for membrane molecular targets

A growing number of RNA aptamers have been selected experimentally using the SELEX combinatorial approach, and these aptamers have several advantages over monoclonal protein antibodies or peptides with respect to their applications in medicine and nanobiotechnology. Relatively few successful selections have been reported for membrane molecular targets, in contrast to the situation with non-membrane molecular targets. This review compares the procedures and techniques used in selections against membrane proteins and membrane lipids. In the case of membrane proteins, the selections were performed against soluble protein fragments, detergent-membrane protein mixed micelles, whole cells, vesicles derived from cellular membranes, and enveloped viruses. Liposomes were used as an experimental system for the selection of aptamers against membrane lipids. RNA structure-dependent aptamer binding for rafts in lipid vesicles was reported. Based on the selected aptamers against DOPC and the amino acid tryptophan, a specific passive membrane transporter composed of RNA was constructed. The determination of the selectivity of aptamers appears to be a crucial step in a selection, but has rarely been fully investigated. The selections, which use whole cells or vesicles derived from membranes, can yield aptamers not only against proteins but also against membrane lipids.     

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.     

前列腺癌适配体的研究进展及其应用

寻找一种高特异性高灵敏度的方法对于前列腺癌的早期发现和及早干预,进而提高患者的生存率及生活质量十分重要。适配体(Aptamer)是由一段寡聚核苷酸链折叠形成的特定三维结构,能高亲和性、高特异性地靶向不同的分子。自上世纪90年代,许多研究者致力于适配体的研究,目前,适配体在药物递送、肿瘤诊疗等方面的研究已取得较大进展。本文将对前列腺癌适配体新近的研究进展及其应用进行综述,以期为将来进一步的研究和临床应用提供参考。     

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.     

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.     

Dimerization of an aptamer generated from Ligand-guided selection (LIGS) yields a high affinity scaffold against B-cells

Nucleic Acid Aptamers (NAAs) are a class of synthetic DNA or RNA molecules that bind specifically to their target. We recently introduced an aptamer termed R1.2 against membrane Immunoglobulin M (mIgM) expressing B-cell neoplasms using Ligand Guided Selection (LIGS). While LIGS-generated aptamers are highly specific, their lower affinity prevents aptamers from being used for translational applications. Highly specific aptamers with higher affinity can increase targetability, boosting the application of aptamers as diagnostic and therapeutic molecules. Herein, we report that dimerization of R1.2, an aptamer generated from LIGS, leads to high affinity variants without compromising the specificity. Three dimeric aptamer analogues with variable linker lengths were designed to evaluate the effect of linker length in affinity. The optimized dimeric R1.2 against cultured B-cell neoplasms, four donor B-cell samples and mIgM-positive Waldenström's Macroglobulinemia (WM) showed specificity. Furthermore, confocal imaging of dimeric aptamer and anti-IgM antibody in purified B-cells suggests co-localization. Binding assays against IgM knockout Burkitt's Lymphoma cells utilizing CRISPR/Cas9 further validated specificity of dimeric R1.2. Collectively, our findings show that LIGS-generated aptamers can be re-engineered into dimeric aptamers with high specificity and affinity, demonstrating wide-range of applicability of LIGS in developing clinically practical diagnostic and therapeutic aptamers.     

Aptamers directly radiolabeled with technetium-99m as a potential agent capable of identifying carcinoembryonic antigen (CEA) in tumor cells T84

Aptamers are small oligonucleotides that are selected to bind with high affinity and specificity to a target molecule. Aptamers are emerging as a new class of molecules for radiopharmaceutical development. In this study a new method to radiolabel aptamers with technetium-99m (^99mTc) was developed. Two aptamers (Apt3 and Apt3-amine) selected against the carcinoembryonic antigen (CEA) were used. Labeling was done by the direct method and the developed complex was subjected to quality control tests. Radiochemical purity and stability were monitored by Thin Layer Chromatography. Binding and specificity assays were carried out in the T84 cell line (CEA+) to evaluate tumor affinity and specificity after radiolabeling. Aptamers were successfully labeled with ^99mTc in high radiochemical yields, showing in vitro stability in presence of plasma and cystein. In binding assays the radiolabeled aptamer Apt3-amine showed the highest affinity to T84 cells. When evaluated with HeLa cells (CEA−), lower uptake was observed, suggesting high specificity for this aptamer. These results suggest that the Apt3-amine aptamer directly labeled with ^99mTc could be considered a promising agent capable of identifying the carcinoembryonic antigen (CEA) present in tumor cells.     

Aptamers for pharmaceuticals and their application in environmental analytics

Aptamers are single-stranded DNA or RNA oligonucleotides, which are able to bind with high affinity and specificity to their target. This property is used for a multitude of applications, for instance as molecular recognition elements in biosensors and other assays. Biosensor application of aptamers offers the possibility for fast and easy detection of environmental relevant substances. Pharmaceutical residues, deriving from human or animal medical treatment, are found in surface, ground, and drinking water. At least the whole range of frequently administered drugs can be detected in noticeable concentrations. Biosensors and assays based on aptamers as specific recognition elements are very convenient for this application because aptamer development is possible for toxic targets. Commonly used biological receptors for biosensors like enzymes or antibodies are mostly unavailable for the detection of pharmaceuticals. This review describes the research activities of aptamer and sensor developments for pharmaceutical detection, with focus on environmental 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.     

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.     

Generation of RNA aptamers to the G-protein-coupled receptor for neurotensin,NTS-1

G-protein-coupled receptors (GPCRs) are integral membrane proteins involved in signal transduction and constitute major drug targets for disease therapy. Aptamers, which are globular RNA or DNA molecules evolved to specifically bind a target, could represent a valuable tool with which to probe the role of such receptors in normal tissue and disease pathology and for cocrystallization with receptors for structure determination by X-ray crystallography. Using the bacterially expressed rat neurotensin receptor NTS-1 as an example, we describe a strategy for the generation of GPCR-specific RNA aptamers. Seven rounds of a "subtractive," paramagnetic bead-based selection protocol were used to enrich for neurotensin receptor-specific aptamers, while circumventing the evolution of aptamers reactive to minor protein contaminants. Representatives of each aptamer family were analyzed in Escherichia coli membrane nitrocellulose filter binding assays. Eight aptamers demonstrated specificity for the neurotensin receptor. One aptamer, P19, was characterized in detail and shown to bind to both the rat receptor and the human receptor with nanomolar affinity. P19 was also shown to interact with rat neurotensin receptor expressed in CHO cells, in both membrane preparations and intact cells. P19 represents the first example of a GPCR-specific RNA aptamer.