Nucleic Acid Aptamers: New Methods for Selection,Stabilization, and Application in Biomedical Science

The adoption of oligonucleotide aptamer is well on the rise, serving an ever increasing demand for versatility in biomedical field. Through the SELEX (Systematic Evolution of Ligands by EXponential enrichment), aptamer that can bind to specific target with high affinity and specificity can be obtained. Aptamers are single-stranded nucleic acid molecules that can fold into complex threedimensional structures, forming binding pockets and clefts for the specific recognition and tight binding of any given molecular target. Recently, aptamers have attracted much attention because they not only have all of the advantages of antibodies, but also have unique merits such as thermal stability, ease of synthesis, reversibility, and little immunogenicity. The advent of novel technologies is revolutionizing aptamer applications. Aptamers can be easily modified by various chemical reactions to introduce functional groups and/or nucleotide extensions. They can also be conjugated to therapeutic molecules such as drugs, drug containing carriers, toxins, or photosensitizers. Here, we discuss new SELEX strategies and stabilization methods as well as applications in drug delivery and molecular imaging.     

Aptamers: new arrows to target dendritic cells

Aptamers, as a novel class of molecular probes for diagnosis, imaging and targeting therapy, have attracted increasing attention in recent years. Aptamers are generated from libraries of single-stranded nucleic acids against different molecules via the “systematic evolution of ligands by exponential enrichment” (SELEX) method. SELEX is a repetitive process of a sequential selection procedure in which a DNA or RNA library pool is incubated separately with target and control molecules to select specific oligonucleotide aptamers with high affinities and specificities. Cell-SELEX is a modified version of the SELEX process in which whole living cells are used as targets for the aptamers. Dendritic cell (DC) targeting, as a new therapeutic approach, can improve the efficiency of immunotherapy in the treatment of allergies and cancers. DCs use various receptors to continuously induce adaptive immunity via capture and presentation of antigens to naïve T cells. DCs are considered as the best targets in modulating immune responses against cancer, autoimmunity, allergy and transplantation. Aptamers, as a new agent, can be applied in DC targeting. The purpose of this review is to present some general concepts of aptamer production and DC targeting by aptamer molecules.     

Recent developments in protein and cell-targeted aptamer selection and applications

Because of their easily modified chemical structures and wide range of targets, aptamers are ideal candidates for various applications, such as biomarker discovery, target diagnosis, molecular imaging, and drug delivery. Aptamers are oligonucleotide sequences that can bind to their targets specifically via unique three dimensional (3-D) structures. Usually, aptamers are obtained from repeated rounds of in vitro or in vivo selection termed SELEX (Systematic Evolution of Ligands by EXponential enrichment), which can generate aptamers with high affinity and specificity for many kinds of targets, such as biomedically important proteins and even cancer cells. In this review, some basic principles and recent developments in the design of SELEX process are discussed, hopefully to provide some guidelines towards performing more efficient aptamer isolation procedures. Moreover, the biomedical and bioanalytical applications of aptamers are further reviewed, based on some smart biochemical modifications of these oligonucleotide structures.     

Cell-based aptamer selection for diagnosing cancer and predicting cancer progression

Antibodies are excellent molecular recognition agents for a wide range of applications therefore they have been used heavily in clinical assays such as disease diagnosis. More recently, aptamers have emerged as alternative capturing agents in a variety of applications including medical diagnosis, environmental toxicity detection, targeted drug delivery and viral therapeutics. Aptamers are ssDNA or RNA that form three dimensional structures and bind to the target molecules such as peptide, protein or small molecules. Aptamers are generated by in vitro process called “SELEX (Systematic Evolution of Ligands by Exponential Enrichment)”. Conventionally, SELEX is performed with immobilized target molecules such as proteins in column, filter or beads. However, for some targets like membrane proteins, it is very difficult or almost impossible to immobilize the target proteins in their active conformation. However, cell-based aptamer selection technology explain how it can be better than standard immobilization methods in brief. Here, we described the cell-specific aptamers selecting technology, called cell-based SELEX, for diagnosing disease and predicting disease progression, especially in the case of complex disease, like cancer.     

Derivation of RNA aptamer inhibitors of human complement C5.

Specific aptamer inhibitors of the human complement C5 component were produced by the SELEX methodology of directed evolution of nucleic acid ligands. The SELEX procedure started with a pool of random-sequence, 2'F-pyrimidine-modified nuclease-stabilized RNA, and after twelve rounds of iterative C5 binding and nucleic acid amplification an evolved RNA pool was obtained which contained the highest affinity binders to the C5 protein. The evolved RNA pool was then cloned and sequenced, and individual clones were analyzed for binding and function. Twenty-eight clones (out of sixty) were identified which bound C5 (termed aptamers). Seven of these aptamers formed a closely related sequence homology family; these aptamers bound C5 with a Kd 20-40 nM and also inhibited human serum hemolytic activity. In addition, these aptamers inhibited zymosan-induced generation of C5a. Aptamer inhibition of both C5b and C5a suggests that aptamer binding inhibits cleavage of C5 by the C5 convertase of both pathways. One of the inhibitory aptamer sequences was truncated to yield a 38-mer 2'F RNA aptamer which retained C5 binding and inhibitory activity. The structure of this aptamer is predicted to be a stem-loop containing thirteen base pairs, and also containing two bulges. The affinity of this aptamer was improved by performing a second biased SELEX experiment, where the randomized starting RNA pool uses a template where the individual base compositions are biased toward a specific sequence. This second SELEX experiment produced an aptamer with a Kd of 2-5 nM which retained functional activity. Another SELEX to rat C5 produced an aptamer with binding and inhibitory properties virtually identical with the human aptamer. The human and rat aptamers are being evaluated for complement inhibition in vitro and in vivo as potential therapeutics for treatment of human disease.     

Aptamers: selection, modification and application to nervous system diseases

Aptamers are nonnaturally occurring oligonucleotides generated by the SELEX (Systematic Evolution of Ligands by Exponential enrichment) process. Due to their unique three-dimensional structures, aptamers can bind to various targets, ranging from small compounds to cells and tissues, with high affinity and specificity. While first reported in 1990, aptamers have become useful tools in the biomedical field because of their unique characteristics, such as easy and quick preparation, cost-effectiveness, small size, versatility, et al. Recently various chemical modifications have been introduced to enhance aptamers' stability in the body fluids and their bioavailability in animals, which have pushed aptamer closer to therapeutic and diagnostic application. This review provides an overview of the aptamer modifications and their application in the nervous system disorders.     

Aptasensor for environmental monitoring

Aptamers are single-stranded oligonucleotides (DNA or RNA) capable of folding into well-defined structures and motifs that allow them bind to various target molecules with high specificity and affinity. The crucial ability of aptamers is interaction with a given target from small ions to molecular level that makes them useful in many applications for specific detection of various analytes. Nowadays aptamer is an effective tool not only for in vitro research in diagnostic methods, drug delivery and treatment therapy, but also for the development of nucleic-acid-based sensors in industrial waste management, pollution control and environmental toxicology. This mini review is focused on discussion and updating about the global researches in nucleic-acid-based sensors for environmental issues.     

Derivatizations of Sgc8‐c aptamer to prepare metallic radiopharmaceuticals as imaging diagnostic agents: Syntheses,isolations, and physicochemical characterizations

Aptamers, oligonucleotides with the capability to bind to a target through non‐covalent bonds with high affinity and specificity, have a great number of advantages as scaffold to prepare molecular imaging agents. In this sense, we have performed post‐SELEX modifications of a truncated aptamer, Sgc8‐c, which bind to protein tyrosine kinase 7 to obtain a specific molecular targeting probe for in vivo diagnosis and in vivo therapy. Herein, we describe the synthetic efforts to prepare conjugates between Sgc8‐c and different metallic ions chelator moieties in short times, high purities, and adequate yields. The selected chelator moieties, derived from 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid, 2‐benzyl‐1,4,7‐triazacyclononane‐1,4,7‐triacetic acid, and 6‐hydrazinonicotinic acid, were covalently attached at the 5′‐aptamer position yielding the expected products which were stable in aqueous solution up to 75°C and in typical aptamer storage conditions at least for 30 days.     

RNA aptamers as potential pharmaceuticals against infections with African trypanosomes

Protozoal pathogens cause symptomatic as well as asymptomatic infections. They have a worldwide impact, which in part is reflected in the long-standing search for antiprotozoal chemotherapy. Unfortunately, effective treatments for the different diseases are by and large not available. This is especially true for African trypanosomiasis, also known as sleeping sickness. The disease is an increasing problem in many parts of sub-Saharan Africa, which is due to the lack of new therapeutics and the increasing resistance against traditional drugs such as melarsoprol, berenil and isometamidium. 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 recognize ligands of different chemical origin. Aptamers typically bind their target with high affinity and high specificity and have successfully been converted into pharmaceutically active compounds. Here we summarize the recent examples of the SELEX technique within the context of identifying high-affinity RNA ligands against the surface of the protozoan parasite Trypanosoma brucei, which is the causative agent of sleeping sickness.     

Aptamers: potential applications to pancreatic cancer therapy

There is an unquestionable need for more effective therapies for pancreatic cancer. Aptamers are single-stranded DNA or RNA oligonucleotide ligands whose 3-dimensional structures are dictated by their sequences. Aptamers have been generated against numerous purified protein targets using an iterative in vitro selection technique known as Systematic Evolution of Ligands by EXponential enrichment (SELEX). Several biochemical properties make them attractive tools for use in an array of biological research applications and as potential pharmacologic agents. Isolated aptamers may directly affect target protein function, or they may also be modified for use as delivery agents for other therapeutic cargo or as imaging agents. More complex selections, using whole cancer cells or tumor tissue, may simultaneously identify novel or unexpected targets and aptamers to inhibit them. This review summarizes recent advances in the field of aptamers and discusses aptamer targets that have relevance to pancreatic cancer.     

Low efficient immobilization of active chloramphenicol for SELEX

Chloramphenicol is an antibacterial antibiotic which interferes with the protein synthesis of microorganisms. However, the use of chloramphenicol should be limited in humans and food products, because it is known to have side effects such as genotoxicity and aplastic anemia in humans. Therefore, it is important to monitor the amount of chloramphenicol in food products. Instead of using conventional analytical methods or antibodies, using aptamers can be a good alternative for measuring the amount of chloramphenicol in food. Aptamers are nucleic acids within 100 base pairs that can bind to target with high specificities and sensitivities, and are derived through a process called SELEX. Here, we report that the immobilization efficiency of chloramphenicol is low with SELEX, which can lead to the low amount of active chloramphenicol immobilized to epoxy resin. This result may be applicable to general SELEX process, especially for negative SELEX during which aptamers that are not bound to targets are removed to increase the amount of aptamers that can bind specifically to targets.     

Noble metal nanomaterial-based aptasensors for microbial toxin detection

Microbial toxins generated by bacteria, fungi and algae cause serious food-safety problems due to the frequent contamination of foodstuffs and their poisonous nature. Becoming acquainted with the contamination condition of foodstuffs is highly dependent on developing sensitive, specific, and accurate methods for targeting microbial toxins. Aptamers, obtained from systematic evolution of ligands by exponential enrichment (SELEX), have significant advantages for microbial toxin analysis, such as small size, reproducible chemical synthesis, and modification, as well as high binding affinity, specificity, and stability. Besides, aptamers have a predictable structure and can be tailored using biomolecular tools (e.g., ligase, endonuclease, exonuclease, polymerase, and so on), which is conducive to the development of flexible and variable amplification methods. Recent studies revealed that the combination of aptamers and noble metal nanomaterials offers unprecedented opportunities for microbial toxin detection. Noble metal nanomaterials with outstanding physical and chemical properties facilitate the detection process and improve the sensitivity and specificity. In this review, we discuss current progress in the development of various noble metal nanomaterial-based aptasensors for microbial toxin detection. These noble metal nanomaterials include gold nanoparticles, gold nanorods, gold nanoclusters, silver nanoparticles, silver nanoclusters, and bimetallic nanomaterials. Aptasensors based on noble metal nanomaterials exhibiting high selectivity and sensitivity represent a promising tool for microbial toxin detection.     

Nucleic acid aptamers: clinical applications and promising new horizons

Aptamers are a special class of nucleic acid molecules that are beginning to be investigated for clinical use. These small RNA/DNA molecules can form secondary and tertiary structures capable of specifically binding proteins or other cellular targets; they are essentially a chemical equivalent of antibodies. Aptamers have the advantage of being highly specific, relatively small in size, and non-immunogenic. Since the discovery of aptamers in the early 1990s, great efforts have been made to make them clinically relevant for diseases like cancer, HIV, and macular degeneration. In the last two decades, many aptamers have been clinically developed as inhibitors for targets such as vascular endothelial growth factor (VEGF) and thrombin. The first aptamer based therapeutic was FDA approved in 2004 for the treatment of age-related macular degeneration and several other aptamers are currently being evaluated in clinical trials. With advances in targeted-therapy, imaging, and nanotechnology, aptamers are readily considered as potential targeting ligands because of their chemical synthesis and ease of modification for conjugation. Preclinical studies using aptamer-siRNA chimeras and aptamer targeted nanoparticle therapeutics have been very successful in mouse models of cancer and HIV. In summary aptamers are in several stages of development, from pre-clinical studies to clinical trials and even as FDA approved therapeutics. In this review, we will discuss the current state of aptamers in clinical trials as well as some promising aptamers in pre-clinical development.     

核酸适配体在抗肿瘤药物主动靶向传递中的应用

核酸适配体是通过指数级富集的配体系统进化技术(systematic evolution of ligands by exponential enrichment,SELEX)从合成的大容量单链随机寡核苷酸文库中筛选并富集的对某些靶点具有高特异性和高结合率的小分子DNA或RNA片段.核酸适配体具有直接抑制肿瘤细胞增殖的作用,通过结构修饰可增强在体内的稳定性.此外,它可与载药纳米结构键合,用于肿瘤诊断和治疗.本文综述了核酸适配体的肿瘤靶点、筛选方法及其在肿瘤诊断和治疗中的应用.     

Anti-HIV inhibitors based on nucleic acids: emergence of aptamers as potent antivirals

The development of resistance and the inability of currently approved antiretroviral drugs to completely eradicate HIV-1 have led to increased focus on therapies other than small molecules. Although nucleic acid-based intervention requires complex tasks involving intracellular delivery and/or stable expression in target cells, recent advances in gene therapy methods combined with continued progress in stem cell approaches have made nucleic acid-based compounds excellent candidates for effectively inhibiting intracellular targets. Consequently, multiple nucleic acid-based therapies are being developed. These include antisense nucleic acids, peptide nucleic acids and RNA decoys, which can interfere with HIV-1 replication. More recently, RNA interference, which exploits a novel cellular pathway, has been shown to effectively reduce viral titers in cell culture and promises to be a potential candidate for suppressing HIV replication in vivo. A promising candidate in the midst of these emerging approaches is the aptamer approach, which involves the use of a class of small nucleic acid molecules isolated from combinatorial libraries by an in vitro evolution protocol termed SELEX. Aptamers exhibit exquisite specificity, high affinity and the virtual lack of immunogenicity, features that make them exceptionally well-suited to combat HIV without affecting the host. The powerful nature of these specific antagonists of protein function could lead to the development of an effective anti-HIV therapy. Several highly specific, nucleic acid aptamers targeting select HIV proteins have been described. Investigations with anti-HIV RNA aptamers have shown an effective block to viral replication. This review summarizes the existing nucleic-acid based approaches to block HIV replication and attempts to chart the current progress in the development of aptamers against HIV, their use in inhibiting the virus replication, prospects for their use in the clinic and potential drawbacks.     

Application of aptamers in therapeutics and for small-molecule detection

Nucleic acids that can bind with high affinity and specificity to target molecules are called "apta mers". Aptamers recognise a large variety of different molecule classes. The main focus of this chapter is small molecules as targets. Aptamers are applied complementarily to antibody technologies and can substitute antibodies or small molecules wherever their different properties, such as biochemical nature or highly discriminating capacities, are advantageous. Examples of promising applications of these versatile molecules are discussed in the field of therapeutics and biotechnology with a special view to small-molecule detection.     

基于SELEX技术的疾病标志物发现新策略研究进展

生物标志物(Biomarker)是指可以标记生物体结构或功能改变的生化指标.在疾病研究中,生物标志物可供客观测定和评价机体所处的生理或病理状态,为疾病的诊断、鉴定提供辅助手段.指数富集的配体系统进化(Systematic evolution of ligands by exponential enrichment,SE...     

ATP-Responsive Drug Delivery Systems

The combination of targeted drug delivery and controlled-release technology may pave the road for more effective yet safer chemotherapeutic options for cancer therapy. Drug-encapsulated polymeric nanoparticle–aptamer bioconjugates represent an emerging technology that can facilitate the delivery of chemotherapeutics to primary and metastatic tumours. Aptamers are short nucleic acid molecules with binding properties and biochemical characteristics that may make them suitable for use as targeting molecules. The goal of this review is to summarise the key components that are required for creating effective cancer targeting nanoparticle–aptamer bioconjugates. The field of controlled release and the structure and properties of aptamers, as well as the criteria for constructing effective conjugates, will be discussed.     

Therapeutic applications of aptamers

Aptamers constitute a new class of oligonucleotides that have gained therapeutic importance. With the approval of the first aptamer drug, pegaptanib, interest in this class of oligonucleotides, often referred to as 'chemical antibodies', has increased. This article discusses aptamers in relation to other oligonucleotide molecules such as antisense nucleotides, short inhibitory sequences, ribozymes and so on. The development of pegaptanib is looked at from the point of view of the challenges faced in converting aptamers into therapeutic molecules. Cases of other aptamers, which show promise as drugs, are discussed in slightly greater detail. Comparison with antibodies and small molecules, which have hitherto held monopoly in this area, is also made.     

Aptamers as inhibitors of target proteins

Background: Aptamers as inhibitors of proteins in therapeutic applications offer great advantages over their antibody counterparts and the promise to be developed into the next generation therapeutic agents. However, the control of aptamer intellectual property (IP) by two major players has made aptamers an area difficult to operate and often off-putting for academic and commercial organisations. Yet, their great potential is keeping aptamers at the research forefront, with one aptamer in the clinic and various at different stages of clinical trials. Objective: To provide a comprehensive review of the aptamer IP landscape and the issues associated with aptamer therapeutics against protein targets. Methods: Extensive review of the scientific and patent literature. Conclusions: Following our experience in developing, patenting and commercialising our aptamers against MUC1 and an extensive review of the literature, we have identified a variety of issues pertaining to the development of aptamers against protein targets for therapeutic applications, their patenting and granting of patents, the original IP holders and their policy, as well as the current market and traits. Despite a slow start, aptamers have been developed against various therapeutic proteins and offer the promise of providing a novel generation of therapeutic entities with a variety of applications.