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.     

Low efficient generation of ssDNA aptamer using chemical modified spacer primer

Aptamers are oligonucleotides (ssDNA or RNA) with an appropriate size of 100 bps that bind with high affinity and specificity to a wide range of target molecules, including virtually any class of protein, drugs or small organic/inorganic molecules. The in vitro selection process referred to as SELEX provides a powerful tool to identify specific aptamers with high affinity and even discriminate between closely related targets. Aptamers have various applications such as analytical tools, disease diagnosis and prediction, pharmaceutical research, drug development, therapy and even for environmental monitoring. Nowadays, with the development of SELEX methods, generation of aptamer becomes more efficient, less time consuming and even automatically. The whole SELEX process includes binding, separation, and nucleic acid amplification. As amplification of nucleotides is an important process in successive SELEX, we will compare several methods for generation of aptamer in this report.     

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.     

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.     

Aptamers as therapeutics in cardiovascular diseases

With many advantages over other therapeutic agents such as monoclonal antibodies, aptamers have recently emerged as a novel and powerful class of ligands with excellent potential for diagnostic and therapeutic applications. Typically generated through Systematic Evolution of Ligands by EXponential enrichment (SELEX), aptamers have been selected against a wide range of targets such as proteins, phospholipids, sugars, nucleic acids, as well as whole cells. DNA/RNA aptamers are single-stranded DNA/RNA oligonucleotides (with a molecular weight of 5-40 kDa) that can fold into well-defined 3D structures and bind to their target molecules with high affinity and specificity. A number of strategies have been adopted to synthesize aptamers with enhanced in vitro/in vivo stability, aiming at potential therapeutic/diagnostic applications in the clinic. In cardiovascular diseases, aptamers can be developed into therapeutic agents as anti-thrombotics, anti-coagulants, among others. This review focuses on aptamers that were selected against various molecular targets involved in cardiovascular diseases: von Willebrand factor (vWF), thrombin, factor IX, phospholamban, P-selectin, platelet-derived growth factor, integrin α(v)β(3), CXCL10, vasopressin, among others. With continued effort in the development of aptamer-based therapeutics, aptamers will find their niches in cardiovascular diseases and significantly impact clinical patient management.     

RNA aptamers: from basic science towards therapy

The SELEX technique (systematic evolution of ligands by exponential enrichment) provides a powerful tool for the in vitro selection of nucleic acid ligands (aptamers) from combinatorial oligonucleotide libraries against a target molecule. In the beginning of the technique's use, RNA molecules were identified that bind to proteins that naturally interact with nucleic acids or to small organic molecules. In the following years, the use of the SELEX technique was extended to isolate oligonucleotide ligands (aptamers) for a wide range of proteins of importance for therapy and diagnostics, such as growth factors and cell surface antigens. These oligonucleotides bind their targets with similar affinities and specificities as antibodies do. The in vitro selection of oligonucleotides with enzymatic activity, denominated aptazymes, allows the direct transduction of molecular recognition to catalysis. Recently, the use of in vitro selection methods to isolate protein inhibitors has been extended to complex targets, such as membrane-bound receptors, and even entire cells. RNA aptamers have also been expressed in living cells. These aptamers, also called intramers, can be used to dissect intracellular signal transduction pathways. The utility of RNA aptamers for in vivo experiments, as well as for diagnostic and therapeutic purposes, is considerably enhanced by chemical modifications, such as substitutions of the 2'-OH groups of the ribose backbone in order to provide resistance against enzymatic degradation in biological fluids. In an alternative approach, Spiegelmers are identified through in vitro selection of an unmodified D-RNA molecule against a mirror-image (i.e. a D-peptide) of a selection target, followed by synthesis of the unnatural nuclease-resistant L-configuration of the RNA aptamer that recognizes the natural configuration of its selection target (i.e. a L-peptide). Recently, nuclease-resistant inhibitory RNA aptamers have been developed against a great variety of targets implicated in disease. Some results have already been obtained in animal models and in clinical trials.     

Peptide aptamers as guides for small-molecule drug discovery

Peptide aptamers are combinatorial protein reagents that bind to target proteins with a high specificity and a strong affinity. By so doing, they can modulate the function of their cognate targets. Because peptide aptamers introduce perturbations that are similar to those caused by therapeutic molecules, their use identifies and/or validates therapeutic targets with a higher confidence level than is typically provided by methods that act upon protein expression levels. The unbiased combinatorial nature of peptide aptamers enables them to 'decorate' numerous polymorphic protein surfaces, whose biological relevance can be inferred through characterization of the peptide aptamers. Bioactive aptamers that bind druggable surfaces can be used in displacement screening assays to identify small-molecule hits to the surfaces. The peptide aptamer technology has a positive impact on drug discovery by addressing major causes of failure and by offering a seamless, cost-effective process from target validation to hit identification.     

The use of synthetic oligonucleotides as protein inhibitors and anticode drugs in cancer therapy: accomplishments and limitations

The function of gene products can be altered at many levels, including the mutation of gene sequence and the change in steady state levels of mRNA and/or protein by various mechanisms. The cumulative malfunction of specific gene products underlies many pathological conditions such as the multi-step and multi-cause acquisition of cancer. Here we discuss two oligonucleotide-based strategies in which these compounds target defective gene products acting either as antiprotein or anticode agents. The SELEX technique (systematic evolution of ligands by exponential enrichment) is an antiprotein approach in which nuclease-resistant DNA or RNA aptamers are selected by their ability to bind their protein targets with high affinity and specificity of the same range as antibodies. Such inhibitors were previously evolved against a great variety of targets, including receptors, growth factors and adhesion molecules implicated in the genesis of some kinds of cancer. Moreover, some results have already been obtained in animal models. The antigene technology interferes with earlier steps in the information flow leading from gene to protein. In this approach selective gene silencing is provided by the formation of stable and specific complexes between triplex forming molecules and their DNA targets. The feasibility of this strategy as well as a molecular mechanism for the action of antigene oligonucleotides has been demonstrated in cellular systems and in vivo. The use of oligonucleotide drugs (of either the antiprotein or the anticode type) as a viable approach to cancer therapy is limited by some common problems that will be discussed.     

Aptamer-based fluorescent biosensors

Selected from random pools of DNA or RNA molecules through systematic evolution of ligands by exponential enrichment (SELEX), aptamers can bind to target molecules with high affinity and specificity, which makes them ideal recognition elements in the development of biosensors. To date, aptamer-based biosensors have used a wide variety of detection techniques, which are briefly summarized in this article. The focus of this review is on the development of aptamer-based fluorescent biosensors, with emphasis on their design as well as properties such as sensitivity and specificity. These biosensors can be broadly divided into two categories: those using fluorescently-labeled aptamers and others that employ label-free aptamers. Within each category, they can be further divided into "signal-on" and "signal-off" sensors. A number of these aptamer-based fluorescent biosensors have shown promising results in biological samples such as urine and serum, suggesting their potential applications in biomedical research and disease diagnostics.     

SELEX技术及寡核苷酸适配体的近期研究进展

指数富集的配体系统进化（SELEX）技术是一类具备蓬勃发展前景的体外筛选技术,在生物学、药学及化学领域已引起广泛关注。本文即针对2004年以来SELEX技术的发展特点,主要介绍两类新型SELEX策略,即毛细管电泳-SELEX和针对复杂靶标的SELEX方法,并简要总结了寡核苷酸适配体在生物医学和药学相关方面的最新应用进展。     

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.     

Aptamers against cell surface receptors: selection, modification and application

Aptamers are synthetic oligonucleotides selected from pools of random-sequence oligonucleotides which bind to a wide range of biomolecular targets with high affinity and specificity. Compared with antibodies, aptamers exhibit significant advantages including small size, easy synthesis and modification, as well as low immunogenicity. Many of the aptamers also show inhibition of their targets, making them potential therapeutic and targeting reagents in clinical applications. Compared with aptamers against intracellular proteins and molecules, however, the identification of aptamers against cell-surface receptors and receptor-related antigens is more difficult, due to the complex cellular environment in which receptors are located, and also the unique conformations and compositions of receptors to keep their activity. In this review, we will introduce the identification, modification and working mechanism of aptamers against cell-surface receptors. Based on the different characteristics of target receptors and selection strategies used, the identified aptamers show distinct binding affinity with recombinant targets or specific cell lines which express receptors on the surface in vitro. Some of the in vivo experiments also indicate that aptamers have the capability of inhibiting the overexpressing receptor-related tumor growth, working as potential anti-tumor therapeutic drugs. Despite of the difficulties during the selection of receptor aptamers and the study of their working mechanism during the present time, it is possible that in the future aptamers will increasingly exhibit therapeutic and diagnostic utility.     

系统进化指数富集法筛选抑制人乙酰胆碱酯酶的RNA分子

目的：获得针对人红细胞乙酰胆碱酯酶(AChE)的特异性核酸配基．方法：利用微孔板法筛选人红细胞膜AChE的核酸配基．利用凝胶阻滞实验检测核酸配基与AChE特异性的结合．微量碱羟胺比色法测定AChE的活性．结果：经过9轮筛选得到针对人红细胞AChE的核酸配基．在相同浓度(2．25μmol/L)下,它们不与人重组丁酰胆碱酯酶(rhBChE)作用,而与人RBCAChE特异结合并抑制AChE的活性．结论：用SELEX技术可以高效地从大容量寡核苷酸组合文库中得到AChE特异性抑制剂．     

Nanoparticle-aptamer bioconjugates for targeted antineoplastic drug delivery

Targeted drug delivery technologies can provide physicians with new approaches to treat and manage patients with cancer. Nucleic acid ligands (aptamers) are a novel class of targeting molecules that can be used in a similar manner to antibodies. Beyond use as drugs themselves, aptamers have the potential to serve as targeting ligands to deliver drugs, imaging agents, or other bioactive agents to the intended site of action. Bioconjugates of nanoparticles and aptamers can selectively bind and be taken up by cancer cells. In this article we review progress to date for antineoplastic drug delivery using nanoparticle-aptamer bioconjugates. Aptamers are isolated through a process of in vitro selection, also referred to as systematic evolution of ligands by exponential enrichment (SELEX). There is an increasing numbers of aptamers for cancer targeting being reported in the literature. These aptamers often interact with antigens that are overexpressed exclusively, or preferentially, on cancer cells or in the cancer microenvironment. As novel drug delivery vehicles, nanoparticle-aptamer bioconjugates may be developed to target a myriad of diseases including many cancers by delivering a variety of therapeutic agents specifically to the site of interest. The first in vivo study of antineoplastic drug delivery by a bioconjugate employed nanoparticle encapsulating docetaxel and aptamers that bind certain prostate cancer cells. In this study using a xenograft murine model of prostate cancer, these bioconjugates were shown to significantly improve tumor reduction after intratumoral injection compared with all controls. Furthermore, the docetaxel-loaded nanoparticle-aptamer bioconjugates demonstrated reduced toxicity in terms of acute bodyweight loss compared with the controls. In vitro, the efficacy of the docetaxel-loaded nanoparticle-aptamer bioconjugate was shown to be due to intracellular delivery of the drug to the cancer cells, and the bioconjugate without the drug had no cytotoxicity. Nanoparticle-aptamer bioconjugates may prove to be useful not only for management of cancer but also various other indications. New aptamers, multivalent targeting strategies, and multimodal treatments such as simultaneous radio- and chemotherapy may further increase the efficacy of these bioconjugates and facilitate their clinical translation for therapeutic and diagnostic applications.     

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...     

Exploring the Utility of ssDNA Aptamers Directed against Snake Venom Toxins as New Therapeutics for Snakebite Envenoming

Snakebite is a neglected tropical disease that causes considerable death and disability in the tropical world. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Antivenoms are the mainstay therapy for treating the toxic effects of snakebite, but despite saving thousands of lives annually, these therapies are associated with limited cross-snake species efficacy due to venom variation, which ultimately restricts their therapeutic utility to particular geographical regions. In this study, we sought to explore the potential of ssDNA aptamers as toxin-specific inhibitory alternatives to antibodies. As a proof of principle model, we selected snake venom serine protease toxins, which are responsible for contributing to venom-induced coagulopathy following snakebite envenoming, as our target. Using SELEX technology, we selected ssDNA aptamers against recombinantly expressed versions of the fibrinogenolytic SVSPs ancrod from the venom of C. rhodostoma and batroxobin from B. atrox. From the resulting pool of specific ssDNA aptamers directed against each target, we identified candidates that exhibited low nanomolar binding affinities to their targets. Downstream aptamer-linked immobilised sorbent assay, fibrinogenolysis, and coagulation profiling experiments demonstrated that the candidate aptamers were able to recognise native and recombinant SVSP toxins and inhibit the toxin- and venom-induced prolongation of plasma clotting times and the consumption of fibrinogen, with inhibitory potencies highly comparable to commercial polyvalent antivenoms. Our findings demonstrate that rationally selected toxin-specific aptamers can exhibit broad in vitro cross-reactivity against toxin isoforms found in different snake venoms and are capable of inhibiting toxins in pathologically relevant in vitro and ex vivo models of venom activity. These data highlight the potential utility of ssDNA aptamers as novel toxin-inhibiting therapeutics of value for tackling snakebite envenoming.     

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.     

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.     

Nucleic acid aptamers against proteases

Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing small molecule protease inhibitors of sufficient specificity has proved a daunting task. Aptamers seem to represent a promising alternative. In our review, we concentrate on biochemical mechanisms of aptamer selection, protein-aptamer recognition, protease inhibition, and advantages of aptamers for pharmacological intervention with pathophysiological functions of proteases. Aptamers can be selected so that they bind their targets highly specifically and with affinities corresponding to KD values in the nM range. Aptamers can be selected so that they recognize their targets conformation-specifically, for instance with vastly different affinities to zymogen and active enzyme forms. Furthermore, aptamers can be selected to inhibit the enzyme activity of the target proteases, but also to inhibit functionally important exosite interactions, for instance cofactor binding. Several protease-inhibiting aptamers, directed against blood coagulation factors, are in clinical trials as anticoagulant drugs. Several of the studies on protease-binding aptamers have been pioneering and trend-setting in the field. The work with protease-binding aptamers also demonstrates many interesting examples of non-standard selection strategies and of new principles for regulating the activity of the inhibitory action of aptamers of general interest to researchers working with nucleic acid aptamers.