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.     

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.     

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.     

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.     

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.     

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.     

Advances on aptamers targeting Plasmodium and trypanosomatids

Aptamers are single-stranded oligonucleotides (ssDNA or RNA) selected from combinatorial libraries by an in vitro process and possess a specific three-dimensional structure depending on its sequence. These molecules are able to recognize and, eventually, alter the activity of their targets by binding directly in a similar way to antibodies. Over the last years, aptamer technology has been used in a wide range of diagnostic and therapeutic applications and, concretely, several strategies are currently being explored using aptamers against Plasmodium and trypanosomatid proteins associated with parasitic diseases which affect hundreds of millions people. One approach tries to block the interaction between the parasite and the host using aptamers targeting host-cell matrix receptors. A second strategy consists in attack the parasite intracellularly targeting heme group or interfering in the intracellular RNA transport. In another strategy, aptamers targeting invariant polypeptides could be used as a specific drug delivery system into the parasite. Finally, aptamers addressed to re-direct the immune response of the infected host are being studied. Other potential use of the aptamers is as biorecognition element in diagnostic systems for parasitic diseases. In this paper, we briefly review how aptamers against Plasmodium and trypanosomatids are discovered, with a focus on recent advances that improve the aptamers properties as a real tool for parasite fighting.     

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.     

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.     

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.     

Potential use of aptamers for diagnosis and treatment of pancreatic cancer

Abstract

Pancreatic cancer (PC) is highly malignant with a low 5-year survival rate. PC currently does not have good early diagnostic markers and responses poorly to chemotherapeutic drugs. The search for better biomarkers and developing more effective chemotherapy are important ways to improve the healthcare of PC patients. Aptamers are single-stranded nucleic acids with high binding affinity and specificity to target molecules. Many aptamers against different forms of cancer including PC have been selected for both diagnostic and therapeutic use. Aptamers can work as ligands to distinguish tumour cells from normal cells. Using cells as selection targets, the obtained aptamers have been used to discover new cancer biomarkers after identification of the binding target. Aptamers have been shown to have antagonists effect on cancer cell proliferation, apoptosis, and metastasis. In addition, aptamers have been used as carriers to deliver therapeutic agents to selectively kill PC cells. This review summarises the potential use of aptamers in the diagnosis and treatment of PC.     

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.     

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.     

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.     

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.     

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 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.     

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.     

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.     

Aptamers: current challenges and future prospects

ABSTRACT

Introduction: Aptamers are oligonucleotide molecules raised in vitro from large combinatorial libraries of nucleic acids and developed to bind to targets with high affinity and specificity. Whereas novel target molecules are proposed for therapeutic intervention and diagnostic, aptamer technology has a great potential to become a source of lead compounds.

Areas covered: In this review, the authors address the current status of the technology and highlight the recent progress in aptamer-based technologies. They also discuss the current major technical limitations of aptamer technology and propose original solutions based on existing technologies that could result in a solid aptamer-discovery platform.

Expert opinion: Whereas aptamers have shown to bind to targets with similar affinities and specificities to those of antibodies, aptamers have several advantages that could outweigh antibody technology and open new opportunities for better medical and diagnostic solutions. However, the current status of the aptamer technology suffers from several technical limitations that slowdown the progression of novel aptamers into the clinic and makes the business around aptamers challenging.