Selection of RNA aptamers against recombinant transforming growth factor-beta type III receptor displayed on cell surface

In most cases, anti-protein aptamers are selected by systematic evolution of ligands by exponential enrichment (SELEX) using purified recombinant protein targets. Cell surface proteins, however, are not easy targets for SELEX due to the difficulties associated with their purification. Here, we developed a novel SELEX procedure (referred to as TECS-SELEX) in which cell-surface displayed recombinant protein is directly used as the selection target. Using this method, we isolated RNA aptamers against transforming growth factor-beta type III receptor expressed on Chinese hamster ovary (CHO) cells. One of the RNA aptamers has a dissociation constant in the 1 nM range and competed with transforming growth factor-beta to bind to the cell surface receptor in vitro.     

Comparison of different strategies to select aptamers against a transmembrane protein target

Binding of aptamers is dependent on their target conformation, which in turn is conditioned by the target's environment. Therefore, selection of aptamers against the active forms of membrane proteins could require their correct membrane insertion in order to maintain their native conformation. Here, we compare different SELEX strategies to identify aptamers against the mutated form of the membrane receptor tyrosine kinase RET(C634Y). (1) selections S1 and S2 against living cells transformed to express the protein yielded a minority of RET-targeted aptamers while the bulk of aptamers recognized more abundant membrane proteins, suggesting that a high level of expression of the target protein is crucial to allow the isolation of aptamers at cell surface; (2) selection S3 against the purified extracellular moiety of RET yielded aptamers unable to recognize RET expressed at the cell membrane; (3) crossover selections S4 and S5 alternating cells and recombinant RET enhanced the enrichment of the aptamers directed against RET; however, these aptamers displayed a weaker affinity for Ret than those obtained with S1 and S2. In our case, using transformed cell lines as the partitioning matrix during SELEX appears to be essential in order to obtain aptamers able to recognize the RET receptor tyrosine kinase in its physiologic environment.     

RNA and DNA aptamers in cytomics analysis.

BACKGROUND: The systematic evolution of ligands by exponential enrichment (SELEX) technique is a combinatorial library approach in which DNA or RNA molecules (aptamers) are selected by their ability to bind their protein targets with high affinity and specificity, comparable to that of monoclonal antibodies. In contrast to antibodies conventionally selected in animals, aptamers are generated by an in vitro selection process, and can be directed against almost every target, including antigens like toxins or nonimmunogenic targets, against which conventional antibodies cannot be raised. METHODS: Aptamers are ideal candidates for cytomics, as they can be attached to fluorescent reporters or nanoparticles in order to study biological function by fluorescence microscopy, by flow cytometry, or to quantify the concentration of their target in biological fluids or cells using ELISA, RIA, and Western blot assays. RESULTS: We demonstrate the in vitro selection of anti-kinin B1 receptor aptamers that could be used to determine B1 receptor expression during inflammation processes. These aptamers specifically recognize their target in a Northern-Western blot assay, and bind to their target protein whenever they are exposed in the membrane. CONCLUSIONS: Currently, aptamers are linked to fluorescent reporters. We discuss here the present status and future directions concerning the use of the SELEX technique in cytomics.     

综述与专论: 核酸适配体筛选与亲和力评价技术主要研究方向、进展与挑战

核酸适配体是一类具有特异性分子识别能力的单链DNA或者RNA分子,通过指数富集的配体系统进化技术（SELEX）筛选得到。核酸适配体相比抗体具有热稳定性高、便于化学合成与修饰、免疫原性低等优点,在生物分析、生物医学、生物技术等众多领域引起广泛关注。高质量的核酸适配体是应用的基础,然而目前能够满足实际应用的核酸适配体数量还非常有限。如何获得高亲和力、高特异性、高体内稳定性的核酸适配体是核酸适配体领域的技术瓶颈。本文首先简单介绍了SELEX技术的基本原理和核酸库的设计、筛选过程监控、次级文库制备、测序和候选适配体筛选等关键步骤。接着归纳总结了30多年来核酸适配体筛选技术的6个主要研究方向、研究进展和局限性。这6个主要研究方向分别是提高适配体特异性的筛选方法、提高适配体稳定性（抗核酸酶降解能力）的筛选方法、快速筛选方法、复杂靶标适配体筛选方法、小分子靶标适配体筛选方法、提高适配体亲和力的筛选方法。其中快速筛选技术是长期以来持续关注的研究方向,几乎所有物理分离手段都已用于提高SELEX的筛选效率。最近,高效化学反应与SELEX技术的结合为核酸适配体的快速筛选提供了新的策略。本文随后对适合小分子靶标核酸适配体筛选的3类方法进展和存在的问题进行了重点评述。这3类方法分别是基于靶标固定的筛选技术、基于文库固定的筛选技术（捕获-SELEX,Capture-SELEX）和均相筛选技术（氧化石墨烯-SELEX,GO-SELEX）。基于靶标固定的筛选技术尽管存在空间位阻等众多问题,由于其操作的简单性,目前依然应用广泛。近年来Capture-SELEX应用广泛。结合36种靶标适配体的筛选实验条件（文库设计、正筛靶标浓度、负筛靶标的选择和浓度）和所获得的适配体的亲和力（K_D,解离常数,dissociation constant）和特异性,对Capture-SELEX的实验条件与适配体性能的关系进行了讨论。统计数据表明,降低正筛靶标浓度有利于提高适配体的亲和力,但不是必要条件。负筛选是目前提高适配体特异性的主要技术手段,但适配体的特异性还不能满足实际需求。负筛选靶标及其浓度的选择差异很大,而且36种靶标中有20种靶标的适配体筛选没有进行负筛选。如何提高核酸适配体的特异性是目前小分子靶标核酸适配体所面临的难题,急需寻找新的策略。本文还列表归纳了近三年利用GO-SELEX进行的13种小分子靶标的实验条件和所获得的适配体的K_D和特异性。统计数据表明,GO-SELEX比Capture-SELEX所需要的筛选轮数少,两种方法所获得的适配体的亲和力多在纳摩尔每升水平。Capture-SELEX相对较低的筛选效率应该主要由于文库的自解离问题。核酸适配体的亲和力评价是候选核酸适配体结构与性能评价的重要组成部分。常用的核酸适配体亲和力评价技术包括基于分离、基于固定、均相体系三大类十多种方法。假阳性和假阴性是各种评价技术都有可能存在的问题。本文以纳米金比色法和等温热滴定技术为例评述技术进展,讨论导致不同亲和力评价技术结果不一致性问题的根本原因。本文最后对核酸适配体筛选技术、亲和力评价技术和技术的标准化的未来发展趋势进行了展望。     

In Vitro Selection of RNA Aptamers Directed Against Protein E: A Haemophilus influenzae Adhesin

Protein E (PE) of Haemophilus influenzae is a highly conserved ubiquitous surface protein involved in adhesion to and activation of epithelial cells. The host proteins—vitronectin, laminin, and plasminogen are major targets for PE-dependent interactions with the host. To identify novel inhibitory molecules of PE, we used an in vitro selection method based on systematic evolution of ligands by exponential enrichment known as SELEX in order to select 2′F-modified RNA aptamers that specifically bind to PE. Fourteen selection cycles were performed with decreasing concentrations of PE. Sequencing of clones from the 14th selection round revealed the presence of semiconserved sequence motifs in loop regions of the RNA aptamers. Among these, three aptamers showed the highest affinity to PE in electrophoretic mobility shift assays and in dot blots. These three aptamers also inhibited the interaction of PE with vitronectin as revealed by ELISA. Moreover, pre-treatment of H. influenzae with the aptamers significantly inhibited binding of vitronectin to the bacterial surface. Biacore experiments indicated that one of the aptamers had a higher binding affinity for PE as compared to the other aptamers. Our results show that it is possible to select RNA inhibitors against bacterial adhesins using SELEX in order to inhibit interactions with target proteins.     

Identification of DNA Aptamers toward Epithelial Cell Adhesion Molecule via Cell-SELEX

The epithelial cell adhesion molecule (EpCAM, also known as CD326) is a transmembrane glycoprotein that is specifically detected in most adenocarcinomas and cancer stem cells. In this study, we performed a Cell systematic evolution of ligands by exponential enrichment (SELEX) experiment to isolate the aptamers against EpCAM. After seven round of Cell SELEX, we identified several aptamer candidates. Among the selected aptamers, EP166 specifically binds to cells expressing EpCAM with an equilibrium dissociation constant (Kd) in a micromolar range. On the other hand, it did not bind to negative control cells. Moreover, EP166 binds to J1ES cells, a mouse embryonic stem cell line. Therefore, the isolated aptamers against EpCAM could be used as a stem cell marker or in other applications in both stem cell and cancer studies.     

以完整细胞为靶子的SELEX技术研究进展

指数富集的配体系统进化（SELEX）是一种从大容量寡核苷酸文库中经反复分离扩增步骤得到针对靶分子的高亲和力、高特异性核酸配基——适配体的体外筛选技术。自1990年以来,SELEX技术得到了迅猛发展,筛选的靶分子已由最初的单一物质发展到完整的动物细胞、细菌病原体等复杂靶子。以完整细胞为靶子的SELEX技术有其独特的技术优势,可以在筛选细胞上特定靶分子未知的情况下进行筛选,为药物筛选、临床诊断、疾病治疗和基础医学研究等带来了新的思路和方法。随着对适配体研究的深入,尤其是纳米材料与其相结合应用,该技术将在肿瘤诊断治疗及微生物检测领域具有更为广泛的应用前景。     

Simultaneous generation of aptamers to multiple gamma-carboxyglutamic acid proteins from a focused aptamer library using DeSELEX and convergent selection

By using the in vitro selection method SELEX against the complex mixture of GLA proteins and utilizing methods to deconvolute the resulting ligands, we were able to successfully generate 2'-ribo purine, 2'-fluoro pyrimidine aptamers to various individual targets in the GLA protein proteome that ranged in concentration from 10 nM to 1.4 microM in plasma. Perhaps not unexpectedly, the majority of the aptamers isolated following SELEX bind the most abundant protein in the mixture, prothrombin (FII), with high affinity. We show that by deselecting the dominant prothrombin aptamer the selection can be redirected. By using this DeSELEX approach, we were able to shift the selection toward other sequences and to less abundant protein targets and obtained an aptamer to Factor IX (FIX). We also demonstrate that by using an RNA library that is focused around a proteome, purified protein targets can then be used to rapidly generate aptamers to the protein targets that are rare in the initial mixture such as Factor VII (FVII) and Factor X (FX). Moreover, for all four proteins targeted (FII, FVII, FIX, and FX), aptamers were identified that could inhibit the individual protein's activitity in coagulation assays. Thus, by applying the concepts of DeSELEX and focused library selection, aptamers specific for any protein in a particular proteome can theoretically be generated, even when the proteins in the mixture are present at very different concentrations.     

Parasite-specific aptamers as biosynthetic reagents and potential pharmaceuticals

Aptamers are short, synthetic nucleic acid molecules. They are generated by a Darwinian-type in vitro evolution method known as 'systematic evolution of ligands by exponential enrichment' (SELEX). SELEX represents an experimental platform to identify rare ligands with predetermined functionality from combinatorial nucleic acid libraries. Since its discovery about 20 years ago the method has been instrumental in identifying a large number of aptamers that recognize targets of very different chemistry and molecular complexity. Although aptamers have been converted into sophisticated biomolecular tools for a diverse set of technologies, only a limited number of aptamers have been selected as binding reagents for parasites or parasite-derived molecules. Here the published examples of aptamers that target Leishmania-, Trypanosoma- and Plasmodia-specific molecules are reviewed.     

Assays for cytokines using aptamers

Aptamers are short nucleic acid sequences that are used as ligands to bind their targets with high affinity. They are generated via the combinatorial chemistry procedure systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have shown much promise towards detection of a variety of protein targets, including cytokines. Specifically, for the determination of cytokines and growth factors, several assays making use of aptamers have been developed, including aptamer-based enzyme-linked immunosorbent assays, antibody-linked oligonucleotide assay, fluorescence (anisotropy and resonance energy transfer) assays, and proximity ligation assays. In this article, the concept of aptamer selection using SELEX and the assay formats using aptamers for the detection of cytokines are discussed.     

Advances in SELEX and application of aptamers in the central nervous system

SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is a screening technique that involves the progressive selection of highly specific ligands by repeated rounds of partition and amplification from a large combinatorial nucleic acid library. The products of the selection are called aptamers, which are short single stranded DNA or RNA molecules, binding with high affinity, attributed to their specific three-dimensional shapes, to a large variety of targets, ranging from small molecules to complex mixtures. Various improvement of the original SELEX method described in 1990 have been obtained recently, such as capillary electrophoresis SELEX, Toggle-SELEX, Tailored-SELEX, Photo-SELEX, and others. These new variants greatly shorten time of selection and improve aptamer affinity and specificity. Such aptamers have great potential as detecting and/or diagnostic reagents. Furthermore, some aptamers specifically inhibit biological functions of targeted proteins, and are considered as potent therapeutic lead structures evaluated in preclinical disease models. Recently, one aptamer has been approved by Food and Drug Administration of US for treating age-related macular degeneration. This review presents recent advances in the field of SELEX with special emphasis on applications of aptamers as analytical, diagnostic and therapeutic tools in the central nervous system.     

SELEX技术及Aptamer研究的新进展

综述了近几年指数富集的配体系统进化(SELEX)技术的改良与寡核苷酸配基(aptamer)研究应用方面的新进展.Aptamer是指利用SELEX(systematicevolutionofligandsbyexponentialenrichment)技术,从随机寡核苷酸文库中筛选获得的能够与靶分子特异结合的短单链寡核苷酸配基,通常具有纳摩尔到皮摩尔的亲和力.高通量筛选的技术特点与aptamer精确识别、易体外合成与修饰等特性,使得aptamer在分析化学与生物医药研究方面具有广阔的应用前景.     

Aptamers Binding to c-Met Inhibiting Tumor Cell Migration

The human receptor tyrosine kinase c-Met plays an important role in the control of critical cellular processes. Since c-Met is frequently over expressed or deregulated in human malignancies, blocking its activation is of special interest for therapy. In normal conditions, the c-Met receptor is activated by its bivalent ligand hepatocyte growth factor (HGF). Also bivalent antibodies can activate the receptor by cross linking, limiting therapeutic applications. We report the generation of the RNA aptamer CLN64 containing 2’-fluoro pyrimidine modifications by systematic evolution of ligands by exponential enrichment (SELEX). CLN64 and a previously described single-stranded DNA (ssDNA) aptamer CLN3 exhibited high specificities and affinities to recombinant and cellular expressed c-Met. Both aptamers effectively inhibited HGF-dependent c-Met activation, signaling and cell migration. We showed that these aptamers did not induce c-Met activation, revealing an advantage over bivalent therapeutic molecules. Both aptamers were shown to bind overlapping epitopes but only CLN3 competed with HGF binding to cMet. In addition to their therapeutic and diagnostic potential, CLN3 and CLN64 aptamers exhibit valuable tools to further understand the structural and functional basis for c-Met activation or inhibition by synthetic ligands and their interplay with HGF binding.     

A novel artificial intelligence-based approach for identification of deoxynucleotide aptamers

The selection of a DNA aptamer through the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method involves multiple binding steps, in which a target and a library of randomized DNA sequences are mixed for selection of a single, nucleotide-specific molecule. Usually, 10 to 20 steps are required for SELEX to be completed. Throughout this process it is necessary to discriminate between true DNA aptamers and unspecified DNA-binding sequences. Thus, a novel machine learning-based approach was developed to support and simplify the early steps of the SELEX process, to help discriminate binding between DNA aptamers from those unspecified targets of DNA-binding sequences. An Artificial Intelligence (AI) approach to identify aptamers were implemented based on Natural Language Processing (NLP) and Machine Learning (ML). NLP method (CountVectorizer) was used to extract information from the nucleotide sequences. Four ML algorithms (Logistic Regression, Decision Tree, Gaussian Naïve Bayes, Support Vector Machines) were trained using data from the NLP method along with sequence information. The best performing model was Support Vector Machines because it had the best ability to discriminate between positive and negative classes. In our model, an Accuracy (A) of 0.995, the fraction of samples that the model correctly classified, and an Area Under the Receiving Operating Curve (AUROC) of 0.998, the degree by which a model is capable of distinguishing between classes, were observed. The developed AI approach is useful to identify potential DNA aptamers to reduce the amount of rounds in a SELEX selection. This new approach could be applied in the design of DNA libraries and result in a more efficient and faster process for DNA aptamers to be chosen during SELEX.     

A sol-gel-based microfluidics system enhances the efficiency of RNA aptamer selection

RNA and DNA aptamers that bind to target molecules with high specificity and affinity have been a focus of diagnostics and therapeutic research. These aptamers are obtained by SELEX often requiring many rounds of selection and amplification. Recently, we have shown the efficient binding and elution of RNA aptamers against target proteins using a microfluidic chip that incorporates 5 sol-gel binding droplets within which specific target proteins are imbedded. Here, we demonstrate that our microfluidic chip in a SELEX experiment greatly improved selection efficiency of RNA aptamers to TATA-binding protein, reducing the number of selection cycles needed to produce high affinity aptamers by about 80%. Many aptamers were identical or homologous to those isolated previously by conventional filter-binding SELEX. The microfluidic chip SELEX is readily scalable using a sol-gel microarray-based target multiplexing. Additionally, we show that sol-gel embedded protein arrays can be used as a high-throughput assay for quantifying binding affinities of aptamers.     

Combining SELEX and the yeast three-hybrid system for in vivo selection and classification of RNA aptamers

Aptamers are small nucleic acid ligands that bind to their targets with specificity and high affinity. They are generated by a combinatorial technology, known as SELEX. This in vitro approach uses iterative cycles of enrichment and amplification to select binders from nucleic acid libraries of high complexity. Here we combine SELEX with the yeast three-hybrid system in order to select for RNA aptamers with in vivo binding activity. As a target molecule, we chose the RNA recognition motif-containing RNA-binding protein Rrm4 from the corn pathogen Ustilago maydis. Rrm4 is an ELAV-like protein containing three N-terminal RNA recognition motifs (RRMs). It has been implicated in microtubule-dependent RNA transport during pathogenic development. After 11 SELEX cycles, four aptamer classes were identified. These sequences were further screened for their in vivo binding activity applying the yeast three-hybrid system. Of the initial aptamer classes only members of two classes were capable of binding in vivo. Testing representatives of both classes against Rrm4 variants mutated in one of the three RRM domains revealed that these aptamers interacted with the third RRM. Thus, the yeast three-hybrid system is a useful extension to the SELEX protocol for the identification and characterization of aptamers with in vivo binding activity.     

Cell-specific aptamer probes for membrane protein elucidation in cancer cells

Disease biomarkers play critical roles in the management of various pathological conditions of diseases. This involves diagnosing diseases, predicting disease progression and monitoring the efficacy of treatment modalities. While efforts to identify specific disease biomarkers using a variety of technologies has increased the number of biomarkers or augmented information about them, the effective use of disease-specific biomarkers is still scarce. Here, we report that a high expression of protein tyrosine kinase 7 (PTK7), a transmembrane receptor protein tyrosine kinase-like molecule, was discovered in a series of leukemia cell lines using whole cell aptamer selection. With the implementation of a two-step strategy (aptamer selection and biomarker discovery), combined with mass spectrometry, PTK7 was ultimately identified as a potential biomarker for T-cell acute lymphoblastic leukemia (T-ALL). Specifically, the aptamers for T-ALL cells were selected using the cell-SELEX process, without any prior knowledge of the cell biomarker population, conjugated with magnetic beads and then used to capture and purify their binding targets on the leukemia cell surface. This demonstrates that a panel of molecular aptamers can be easily generated for a specific type of diseased cells. It further demonstrates that this two-step strategy, that is, first selecting cancer cell-specific aptamers and then identifying their binding target proteins, has major clinical implications in that the technique promises to substantially improve the overall effectiveness of biomarker discovery. Specifically, our strategy will enable efficient discovery of new malignancy-related biomarkers, facilitate the development of diagnostic tools and therapeutic approaches to cancer, and markedly improve our understanding of cancer biology.     

SELEX技术在神经系统疾病研究中的应用

配体指数级富集系统进化（systematic evolution of ligands by exponential enrichment,SELEX）技术是一种组合化学技术,可经过反复筛选扩增得到针对靶分子的高亲和力和高特异性的适配子.适配子通过识别、结合特定靶分子并对其进行功能调控从而达到对疾病诊断和治疗的目的 .近年来SELEX技术在神经系统功能和疾病研究中的应用越来越多.现已经筛选出针对朊蛋白、肌腱蛋白-C、β-淀粉样肽、乙酰胆碱受体的自身抗体等靶标的适配子,促进了对朊病毒病、脑肿瘤、阿茨海默病、重症肌无力等神经系统疾病的诊断和治疗研究,为这些疾病的诊治提供了新的研究工具.     

SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands

SELEX stands for systematic evolution of ligands by exponential enrichment. This method, described primarily in 1990 [Ellington, A.D., Szostak, J.W., 1990. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818-822; Tuerk, C., Gold, L., 1990. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505-510] aims at the development of aptamers, which are oligonucleotides (RNA or ssDNA) binding to their target with high selectivity and sensitivity because of their three-dimensional shape. Aptamers are all new ligands with a high affinity for considerably differing molecules ranging from large targets as proteins over peptides, complex molecules to drugs and organic small molecules or even metal ions. Aptamers are widely used, including medical and pharmaceutical basic research, drug development, diagnosis, and therapy. Analytical and separation tools bearing aptamers as molecular recognition and binding elements are another big field of application. Moreover, aptamers are used for the investigation of binding phenomena in proteomics. The SELEX method was modified over the years in different ways to become more efficient and less time consuming, to reach higher affinities of the aptamers selected and for automation of the process. This review is focused on the development of aptamers by use of SELEX and gives an overview about technologies, advantages, limitations, and applications of aptamers.