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．     

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.     

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.     

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.     

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.     

Improving the stability of aptamers by chemical modification

Ever since the invention of SELEX (systematic evolution of ligands by exponential enrichment), there has been rapid development for aptamers over the last two decades, making them a promising approach in therapeutic applications as either drug candidates or diagnostic tools. For therapeutic purposes, a durable performance of aptamers in biofluids is required, which is, however, hampered by the lack of stability of most aptamers. Not only are the nucleic acid aptamers susceptible to nucleases, the peptide aptamers are also subjective to degradation by proteases. With the advancement of chemical biology, numerous attempts have been made to overcome this obstacle, many resulting in significant improvements in stability. In this review, chemical modifications to increase the stability of three main types of aptamers, DNA, RNA and peptide are comprehensively summarized. For nucleic acid aptamers, development of modified SELEX coupled with mutated polymerase is discussed, which is adaptive to a number of modifications in aptamers and in a large extent facilitates the research of aptamer-modifications. For peptide aptamers, approaches in molecular biology with introduction of stabilizing protein as well as the switch of scaffold protein are included, which may represent a future direction of chemical conjugations to aptamers.     

Aptamers: from bench side research towards patented molecules with therapeutic applications

Background: RNA and DNA aptamers recognize their targets with high specificity and affinity. These aptamers can be developed against almost any target protein through iterative cycles of in vitro screening of a combinatorial oligonucleotide library for target binding. Aptamer sequences from the final pool of in vitro selection are screened for pharmacological activity and possible medical applications. Methods: Chemical modifications and improvements of the identification of aptamer selection procedures made aptamers rival antibodies in diagnostic and therapeutic applications. This article reviews recent literature and patents and discusses the properties of aptamers as high-affinity and specificity target binders as well as their stability in biological fluids that turns them into therapeutic agents. Conclusion: The development of aptamers into compounds with therapeutic and diagnostic compounds has resulted in patents protecting the sequences and the use of these oligonucleotides. Several of these patented aptamers are currently being tested in Phase I or II clinical trials. Moreover, an anti-VEGF aptamer has already been approved by the FDA for treatment of age-related macular degeneration in humans.     

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...     

RNA Aptamer-Based Functional Ligands of the Neurotrophin Receptor, TrkB

Many cell surface signaling receptors, such as the neurotrophin receptor, TrkB, have emerged as potential therapeutic targets for diverse diseases. Reduced activation of TrkB in particular is thought to contribute to neurodegenerative diseases. Unfortunately, development of therapeutic reagents that selectively activate particular cell surface receptors such as TrkB has proven challenging. Like many cell surface signaling receptors, TrkB is internalized upon activation; in this proof-of-concept study, we exploited this fact to isolate a pool of nuclease-stabilized RNA aptamers enriched for TrkB agonists. One of the selected aptamers, C4-3, was characterized with recombinant protein-binding assays, cell-based signaling and functional assays, and, in vivo in a seizure model in mice. C4-3 binds the extracellular domain of TrkB with high affinity (K(D) ～2 nM) and exhibits potent TrkB partial agonistic activity and neuroprotective effects in cultured cortical neurons. In mice, C4-3 activates TrkB upon infusion into the hippocampus; systemic administration of C4-3 potentiates kainic acid-induced seizure development. We conclude that C4-3 is a potentially useful therapeutic agent for neurodegenerative diseases in which reduced TrkB activation has been implicated. We anticipate that the cell-based aptamer selection approach used here will be broadly applicable to the identification of aptamer-based agonists for a variety of cell-surface signaling receptors.     

Characterization of RNA aptamers directed against the nucleocapsid protein of Rift Valley fever virus

Nucleocapsid protein (N) is an essential RNA binding protein in many RNA viruses. During replication, N protein encapsidates viral genomic and antigenomic RNA, but not viral mRNA or other cellular RNAs. To discriminate between different species of RNA in a host cell, it is likely that N interacts with specific sequences and/or secondary structures on its target RNA. In this study, we explore the RNA binding properties of N using both natural and artificially selected RNAs as ligands. We found that N binds to RNAs that resemble the terminal panhandle structures of RVFV genomic and antigenomic RNA. Furthermore, we used SELEX to isolate RNA aptamers that bound N with high affinity and determined that N specifically recognizes and binds to GAUU and pyrimidine/guanine motifs. Interestingly, BLAST analysis revealed the presence of these motifs within the coding region of the viral genome, suggesting that N may interact with non-terminal viral RNA sequences during replication. Finally, the aptamer RNAs were used to construct a sensitive fluorescence based sensor of N binding with potential applications for drug screening and imaging methodologies.     

Selection and Characterization of a Novel DNA Aptamer for Label-Free Fluorescence Biosensing of Ochratoxin A

Nucleic acid aptamers are emerging as useful molecular recognition tools for food safety monitoring. However, practical and technical challenges limit the number and diversity of available aptamer probes that can be incorporated into novel sensing schemes. This work describes the selection of novel DNA aptamers that bind to the important food contaminant ochratoxin A (OTA). Following 15 rounds of in vitro selection, sequences were analyzed for OTA binding. Two of the isolated aptamers demonstrated high affinity binding and selectivity to this mycotoxin compared to similar food adulterants. These sequences, as well as a truncated aptamer (minimal sequence required for binding), were incorporated into a SYBR^® Green I fluorescence-based OTA biosensing scheme. This label-free detection platform is capable of rapid, selective, and sensitive OTA quantification with a limit of detection of 9 nM and linear quantification up to 100 nM.     

Single-stranded DNA aptamer that specifically binds to the influenza virus NS1 protein suppresses interferon antagonism

Non-structural protein 1 (NS1) of the influenza A virus (IAV) inhibits the host’s innate immune response by suppressing the induction of interferons (IFNs). Therefore, blocking NS1 activity can be a potential strategy in the development of antiviral agents against IAV infection. In the present study, we selected a single-stranded DNA aptamer specific to the IAV NS1 protein after 15 cycles of systematic evolution of ligands by exponential enrichment (SELEX) procedure and examined the ability of the selected aptamer to inhibit the function of NS1. The selected aptamer binds to NS1 with a K_d of 18.91 ± 3.95 nM and RNA binding domain of NS1 is determined to be critical for the aptamer binding. The aptamer has a G-rich sequence in the random sequence region and forms a G-quadruplex structure. The localization of the aptamer bound to NS1 in cells was determined by confocal images, and flow cytometry analysis further demonstrated that the selected aptamer binds specifically to NS1. In addition, luciferase reporter gene assay, quantitative RT-PCR, and enzyme-linked immunosorbent assay (ELISA) experiments demonstrated that the selected aptamer had the ability to induce IFN-β by suppressing the function of NS1. Importantly, we also found that the selected aptamer was able to inhibit the viral replication without affecting cell viability. These results indicate that the selected ssDNA aptamer has strong potential to be further developed as a therapeutic agent against IAV.     

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.     

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.     

Development of highly nuclease-resistant chemically-modified oligonucleotides

Chemical modification of therapeutic oligodeoxyribonucleotides (ODNs) is necessary to avoid not only degradation by endo- and exo-nucleases but also recognition by sensors such as an innate immune system. We have been developing modified nucleosides having an aminoalky linker at the pyrimidine nucleobase or sugar moiety. ODNs containing 5-N-(6-aminohexyl)carbamoyl-2'-deoxyuridine (7) were thermally stabilized about 3°C per modification and were about 160 times more stable to hydrolysis by snake venom phosphodiesterase (a 3'-exonuclease) than unmodified ODNs, but not by endonucleases. On the other hand, ODNs containing 4'-C-(aminoethyl)thymidine (14b), which was synthesized by a newly developed radical cyclization-ring-enlargement reaction by us, were 87 times more stable to hydrolysis by DNase I (an endonuclease) and 133 times more stable in 50% human serum than unmodified ODNs. The highly stereoselective synthesis of 4'-thioribonuclesides ((S)Ns) was also developed using a Pummerer reaction. Human thrombin RNA aptamer (CII-1-37) containing 4'-thiouridine and 4'-thiocytidine was obtained by SELEX with a K(d) value of 4.7 nM, while a previously known RNA aptamer (RNA-24) has a K(d) value of 85 nM. Studies of the modification pattern-RNAi activity relationships by using (S)Ns have been carried out against luciferase genes. We found that siRNAs, which have 4 residues of (S)Ns on both ends of the sense strand and 4 residues on the 3'-end of the antisense strand, were the most effective. 4'-ThioRNA is about 1100 times more stable in 50% human plasma than unmodified RNA. However, oligoribonucleotides ((SM)ONs) containing 2'-O-methyl-4'-thioribonucleosides were 9800 times more stable in 50% human plasma than unmodified RNA. Since (SM)ON duplexes were thermally more stable than unmodified ON duplexes, therefore they would be quite suitable to use for oligonucleotide therapeutics.     

G-quadruplex DNA aptamers and their ligands: structure, function and application

Highly specific and tight-binding nucleic acid aptamers have been selected against a variety of molecular targets for over 20 years. A significant proportion of these oligonucleotides display G-quadruplex structures, particularly for DNA aptamers, that enable molecular recognition of their ligands. G-quadruplex structures couple a common scaffold to varying loop motifs that act in target recognition. Here, we review DNA G-quadruplex aptamers and their ligands from a structural and functional perspective. We compare the diversity of DNA G-quadruplex aptamers selected against multiple ligand targets, and consider structure with a particular focus on dissecting the thrombin binding aptamer - thrombin interaction. Therapeutic and analytical applications of DNA G-quadruplex aptamers are also discussed. Understanding DNA G-quadruplex aptamers carries implications not only for therapeutics and diagnostics, but also in the natural biochemistry of guanine-rich nucleic acids.     

High-affinity binding of [3H]6-nitroquipazine to 5-hydroxytryptamine transporter in human platelets

The characteristics of the binding [3H]6-nitroquipazine, a very potent and selective inhibitor of 5-hydroxytryptamine (5-HT; serotonin) uptake, to human platelet membranes were studied at a physiological temperature of 37 degrees C. The presence of a single saturable high-affinity binding component for [3H]6-nitroquipazine was demonstrated Non-specific binding was estimated in the presence of 1 microM paroxetine. Scatchard analysis revealed an apparent equilibrium dissociation constant (Kd) of 0.450 +/- 0.04 nM and a maximal number of binding sites (Bmax) of 2508 +/- 360 fmol/mg protein (mean +/- S.D., n = 4). The kinetically derived dissociation constant (Kd) was 0.431 nM. [3H]6-Nitroquipazine binding was inhibited selectively by 5-HT uptake inhibitors, and the potency of various drugs to inhibit [3H]6-nitroquipazine binding closely correlated with their inhibitory effects on [3H]5-HT uptake into synaptosome. Moreover, Ki values for drug inhibition of [3H]6-nitroquipazine binding to human platelet membranes were significantly correlated with the corresponding Ki values for inhibition of [3H]paroxetine binding at 37 degrees C. The present results suggest that the binding sites for [3H]6-nitroquipazine are associated with the 5-HT transporter in human platelets.     

An improved radiolabelled RNA aptamer molecule for HER2 imaging in cancers

Abstract

Human epidermal growth factor receptor 2 (HER2) expression has been shown to be increased in several types of human tumours. In this study, for the imaging of HER2-related tumours, a modified RNA aptamer with HER2-specific targeting was labelled with ^99mTc, by using hydrazino nicotinamide (HYNIC) as the chelator in the presence of tricine or ethylenediamine-N,N′-diacetic acid (EDDA) as the co-ligand. Stability testing of the radiolabelled aptamers in the serum was performed through SDS-PAGE. The aptamer–radionuclide conjugate was evaluated for its cellular HER2-specific binding in ovarian cancer cells (SKOV-3), and its biodistribution properties were assessed in normal and SKOV-3 tumour-bearing mice. In the presence of either tricine or EDDA, the HYNIC-RNA aptamers were labelled with ^99mTc at a high yield and radiochemical purity. Cellular experiments confirmed the specific binding of the RNA aptamer to the HER2 receptor. In the animal biodistribution study, uptake of the EDDA-co-liganded ^99mTc-HYNIC-RNA aptamer by the liver and spleen was remarkably lower than that of the aptamer with tricine. Tumours also showed a higher accumulation of radioactivity with the EDDA-co-liganded aptamer complex. This study demonstrated EDDA to be better than tricine for use as a co-ligand with the RNA aptamer, which can be a potential tool for the molecular imaging of HER2-overexpressing cancers.     

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.