SELEX技术筛选变形链球菌UA159适配子可行性的研究

目的:研究SELEX技术用于筛选口腔致龋菌适配子的可行性。方法:化学合成长度为35mer的随机ssDNA文库,利用SE-LEX技术,分别以变形链球菌UA159(以下简称变链UA159)、乳杆菌和离心管作为靶物质,筛选适配子,不对称PCR扩增筛选产物,所得适配子进行克隆、测序,分析其二级结构,并对其二级结构进行了初步分析。结果:显示各个靶物质的筛选产物在第二轮筛选时就已经表现出具有特征性的二级结构。结论:SELEX技术可以用于口腔致龋菌适配子的筛选。     

Characterisation of aptamer–target interactions by branched selection and high-throughput sequencing of SELEX pools

Nucleic acid aptamer selection by systematic evolution of ligands by exponential enrichment (SELEX) has shown great promise for use in the development of research tools, therapeutics and diagnostics. Typically, aptamers are identified from libraries containing up to 10¹⁶ different RNA or DNA sequences by 5–10 rounds of affinity selection towards a target of interest. Such library screenings can result in complex pools of many target-binding aptamers. New high-throughput sequencing techniques may potentially revolutionise aptamer selection by allowing quantitative assessment of the dynamic changes in the pool composition during the SELEX process and by facilitating large-scale post-SELEX characterisation. In the present study, we demonstrate how high-throughput sequencing of SELEX pools, before and after a single round of branched selection for binding to different target variants, can provide detailed information about aptamer binding sites, preferences for specific target conformations, and functional effects of the aptamers. The procedure was applied on a diverse pool of 2′-fluoropyrimidine-modified RNA enriched for aptamers specific for the serpin plasminogen activator inhibitor-1 (PAI-1) through five rounds of standard selection. The results demonstrate that it is possible to perform large-scale detailed characterisation of aptamer sequences directly in the complex pools obtained from library selection methods, thus without the need to produce individual aptamers.     

SELEX for tubulin affords specific T-rich DNA aptamers. Systematic evolution of ligands by exponeential enrichment.

We succeeded in acquiring two DNA aptamers that selectively recognize tubulin by the SELEX method. A pool of single-stranded oligo-DNAs including a random region of 59 nucleotides was screened by SELEX for tubulin purified from calf-brain as a target. After 20 repetitions of selection round, the library converged on specific T-rich sequences. The binding activity of T-rich clones was analyzed by the SPR sensor to determine their dissociation constants to be in the order of 10 microM.     

In vitro selection of DNA aptamers to anthrax spores with electrochemiluminescence detection.

Systematic evolution of ligands by exponential enrichment (SELEX) was used to select and PCR amplify DNA sequences (aptamers) capable of binding to and detecting nonpathogenic Sterne strain Bacillus anthracis spores. A simplified affinity separation approach was employed, in which autoclaved anthrax spores were used as the separation matrix. An aptamer-magnetic bead-electrochemiluminescence (AM-ECL) sandwich assay scheme was devised for detecting anthrax spores. Using a low SELEX DNA to spore ratio (154 ng DNA/10(6) spores), at least three distinct populations of single-stranded DNA aptamers, having varied affinities for anthrax spores, were noted by the AM-ECL assay. Results reflect detection of spore components with a dynamic range equivalent to < 10- > 6 x 10(6) anthrax spores. In the low DNA to spore ratio experiments, aptamers could be liberated from spore pellets by heating at 96 degrees C for 5 min after each round of SELEX. When a much higher DNA to spore ratio (10,256 ng DNA/10(6) spores) was used for SELEX development, a higher affinity set of aptamers was selected that could not be heat-eluted even at 99 degrees C for 5 min following round four of SELEX. However, high affinity spore surface bound aptamers were detectable via their 5'-biotinylated tails using labeled avidin and could be eluted in deionized water. Aptamers have potential for use as inexpensive, in vitro-generated receptors for biosensors in biological warfare detection and other areas.     

Selection of a DNA aptamer that binds 8-OHdG using GMP-agarose

DNA aptamers, which bind specific molecule, such as 8-OHdG, with high affinity were investigated using an in vitro selection strategy called systematic evolution of ligands by exponential enrichment (SELEX). However, 8-OHdG was difficult to immobilize on a carrier for SELEX. Therefore, a DNA aptamer binding to 8-OHdG was selected using GMP-agarose as an analogue from a library of about 4⁶⁰ random ssDNA sources. As a result, three aptamer candidates were selected. Among the selected DNA aptamers, the No. 22 DNA aptamer exhibited a high affinity for 8-OHdG. The dissociation constant, K_D, of No. 22 DNA aptamer was on the order of 0.1 μmol/L. This result suggests that using an analogue will be a useful new SELEX method for obtaining various aptamers that are difficult to immobilize on a matrix.     

Monitoring Genomic Sequences during SELEX Using High-Throughput Sequencing: Neutral SELEX

Background

SELEX is a well established in vitro selection tool to analyze the structure of ligand-binding nucleic acid sequences called aptamers. Genomic SELEX transforms SELEX into a tool to discover novel, genomically encoded RNA or DNA sequences binding a ligand of interest, called genomic aptamers. Concerns have been raised regarding requirements imposed on RNA sequences undergoing SELEX selection.

Methodology/Principal Findings

To evaluate SELEX and assess the extent of these effects, we designed and performed a Neutral SELEX experiment omitting the selection step, such that the sequences are under the sole selective pressure of SELEX''s amplification steps. Using high-throughput sequencing, we obtained thousands of full-length sequences from the initial genomic library and the pools after each of the 10 rounds of Neutral SELEX. We compared these to sequences obtained from a Genomic SELEX experiment deriving from the same initial library, but screening for RNAs binding with high affinity to the E. coli regulator protein Hfq. With each round of Neutral SELEX, sequences became less stable and changed in nucleotide content, but no sequences were enriched. In contrast, we detected substantial enrichment in the Hfq-selected set with enriched sequences having structural stability similar to the neutral sequences but with significantly different nucleotide selection.

Conclusions/Significance

Our data indicate that positive selection in SELEX acts independently of the neutral selective requirements imposed on the sequences. We conclude that Genomic SELEX, when combined with high-throughput sequencing of positively and neutrally selected pools, as well as the gnomic library, is a powerful method to identify genomic aptamers.     

抑制肿瘤坏死因子-α的DNA适配子的筛选与鉴定

应用SELEX技术筛选能与TNF结合的DNA适配子。化学合成随机寡聚DNA库,以TNF为靶蛋白,经过12轮SELEX筛选,将所得产物克隆、测序。根据所测序列化学合成寡聚DNA适配子,用生物素_亲和素_辣根过氧化物酶显色系统检测适配子与TNF的结合活性;用鼠L929细胞检测适配子拮抗TNF活性。结果显示,所筛选到的寡聚DNA能与TNF-α高亲和力结合,并能在细胞培养中拮抗TNF-α的细胞毒活性。     

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.     

Single-stranded DNA aptamers that bind differentiated but not parental cells: subtractive systematic evolution of ligands by exponential enrichment

In this paper, single-stranded (ss)DNA aptamers with capability to distinguish differentiated PC12 cells from normal PC12 cells were selected by subtractive systematic evolution of ligands by exponential enrichment (SELEX) method. Before each round of selection, randomized ssDNAs were incubated with regular PC12 cells to eliminate those that recognize the common cellular components of both differentiated and undifferentiated PC12 cells. After six rounds of cell-based selection, both of individual aptamers and aptamers of the sixth round pool were found binding to differentiated PC12 cells, but not to the parental PC12 cells. The aptamers of the starting pool showed no such binding. Sequence analysis illustrated that the amount of G content in central random region of these aptamers was much higher than that of the starting pool, which would be expected to be average. The aptamers obtained from this method were also able to identify differentiated PC12 cells from a mixture of both normal and differentiated cells. The results indicate that subtractive SELEX is a useful tool in finding ligands to specific biological markers that distinguish a subtype of cells from cells of homologous origin, such as carcinoma cells among normal epithelial tissues. Both these aptamers and their markers may play important roles in basic research and clinical diagnosis.     

DNA aptamers that bind to PrP(C) and not PrP(Sc) show sequence and structure specificity

DNA aptamers were selected against recombinant human (rhu) cellular prion protein (PrP(C)) 23-231 by systematic evolution of ligands via a systematic evolution of ligands by exponential (SELEX) enrichment procedure using lateral flow chromatography. The SELEX procedure was performed with an aptamer library consisting of a randomized 40-nucleotide core flanked by 28-mer primer-binding sites that, theoretically, represented approximately 10(24) distinct nucleic acid species. Sixty nanograms of rhuPrP(C)23-231 immobilized in the center of a lateral flow device was used as the target molecule for SELEX. At the end of 6 iterations of SELEX, 13 distinct candidate aptamers were identified, of which, 3 aptamers represented 32%, 8%, and 5% of the sequences respectively. Eight aptamers, including the three most frequently occurring candidates, were selected for further evaluation. Selected aptamers bound to rhuPrP(C)23-231 at 10(-6) M to 10(-8) M concentrations. Two of the eight aptamers bound at higher concentrations to rhuPrP(C)90-231. Theoretical thermodynamic modeling of selected aptamer sequences identified several common motifs among the selected aptamers that could play a role in PrP binding. Binding affinity to rhuPrP(C)23-231 was both aptamer sequence and structure dependent. Further, selected aptamers bound to mammalian PrPs derived from brain of healthy sheep, calf, piglet, and deer, and to PrP(C) expressed in mouse neuroblastoma cells. None of the aptamers bound to proteinase K-digested scrapie-infected mouse neuroblastoma cells or untreated PrP-null cells, which further confirmed the PrP(C) specificity of the aptamers. In summary, we enriched and selected DNA aptamers that bind specifically to rhuPrP(C) and mammalian PrP(C) with varying affinities and can be applied to biological samples for PrP(C) enrichment and as diagnostic tools in double ligand assay systems.     

筛选环孢霉素A适体的SELEX技术的建立

体外合成一个全长78个核苷酸,中间含35个随机序列的随机单链寡核苷酸序列(ssDNA)文库,运用指数富集的配体系统进化(SELEX)技术,以环孢霉素A(CsA)为靶目标,以磁珠作为筛选介质,利用生物素 链酶抗生物素 辣根过氧化物酶系统,检测每轮ssDNA文库与CsA的亲和力,筛选并鉴定CsA特异性的适体.经过11轮的筛选,ssDNA文库与CsA的亲和力呈上升趋势.将第10轮筛选产物克隆测序并运用相关软件进行一级结构和二级结构分析.随机挑选的19个克隆适体,根据一级结构的同源性可分为5个家族,二级结构预测以茎环(发夹)为主,这可能是适体与CsA作用的部位. CsA特异性的适体将用于酶联法、免疫荧光法等对CsA进行检测.     

SELEX技术筛选纤维蛋白适配子的研究

目的:用纤维蛋白作为靶物质对ss DNA随机序列文库进行筛选,旨在获得高亲和力的纤维蛋白适配子。方法:在体外人工合成长度为99个核苷酸的ss DNA随机序列文库,文库中间区域为63个核苷酸的随机序列,两端为18个核苷酸的固定的引物序列;然后以羧基磁珠为介质包被纤维蛋白,利用指数级富集的配体系统进化技术(SELEX)对ss DNA随机序列文库进行反复筛选,当结合率不再提高时对筛选出的适配子进行连接、转化及测序分析。结果:羧基磁珠成功地包被了纤维蛋白,包被效率为87.65%,经15轮逐步递增压力的筛选,获得了纤维蛋白适配子群,经测序分析比对发现适配子有很好的多样性。结论:应用SELEX技术初步筛选出了亲和力较高的纤维蛋白适配子群,为下一步的鉴定及功能研究奠定了良好基础。     

DNA aptamers developed against a soman derivative cross‐react with the methylphosphonic acid core but not with flanking hydrophobic groups

Twelve rounds of systematic evolution of ligands by exponential enrichment (SELEX) were conducted against a magnetic bead conjugate of the para‐aminophenylpinacolylmethylphosphonate (PAPMP) derivative of the organophosphorus (OP) nerve agent soman (GD). The goal was to develop DNA aptamers that could scavenge GD in vivo, thereby reducing or eliminating the toxic effects of this dangerous compound. Aptamers were sequenced and screened in peroxidase‐based colorimetric plate assays after rounds 8 and 12 of SELEX. The aptamer candidate sequences exhibiting the highest affinity for the GD derivative from round 8 also reappeared in several clones from round 12. Each of the highest affinity PAPMP‐binding aptamers also bound methylphosphonic acid (MPA). In addition, the aptamer with the highest overall affinity for PAPMP carried a sequence motif (TTTAGT) thought to bind MPA based on previously published data (J. Fluoresc 18: 867–876, 2008). This sequence motif was found in several other relatively high affinity PAPMP aptamer candidates as well. In studies with the nerve agent GD, pre‐incubation of a large molar excess of aptamer candidates failed to protect human butyrylcholinesterase (BuChE) from inhibition. With the aid of three‐dimensional molecular modeling of the GD derivative it appears that a hydrophilic cleft sandwiched between the pinacolyl group and the p‐aminophenyl ring might channel nucleotide interactions to the phosphonate portion of the immobilized GD derivative. However, bona fide GD free in solution may be repulsed by the negative phosphate backbone of aptamers and rotate its phosphonate and fluorine moieties away from the aptamer to avoid being bound. Future attempts to develop aptamers to GD might benefit from immobilizing the pinacolyl group of bona fide GD to enhance exposure of the phosphonate and fluorine to the random DNA library. Copyright © 2008 John Wiley & Sons, Ltd.     

金黄色葡萄球菌外毒素B特异性适体的筛选及其应用

目的:利用指数富集配基的系统进化(SELEX)技术,筛选能与金黄色葡萄球菌外毒素B(SEB)特异、高亲和力结合的单链DNA(ssDNA)适体,并将该适体应用于患者血清标本的检测。方法:从体外合成的96核苷酸随机ss-DNA文库中,以羧基磁珠作为筛选介质,经逐步PCR扩增、筛选,获得针对SEB的高亲和力、高特异性适体;利用荧光素标记适体测定筛选过程中各轮结合力;利用酶连接适体方法检测适体特异性和结合力。结果:经过13轮筛选,ssDNA文库与SEB的结合百分率从1.1%提高到39.8%,增加了36倍;获得的ssDNA适体(A11)针对SEB的特异性强,与金黄色葡萄球菌表面蛋白A(SPA)结合低,并能初步识别患者血清。结论:利用SELEX技术筛选获得了特异结合SEB的高亲和力的ssDNA适体,为金黄色葡萄球菌的临床诊断与治疗奠定了基础。     

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。     

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.     

用于未纯化蛋白样品核酸适配体筛选的Western印迹-SELEX筛选技术的建立

目的:建立一种基于Western印迹的指数式富集的配体系统进化(SELEX)技术,用于未纯化蛋白样品核酸适配体筛选。方法:将目的蛋白经SDS-PAGE分离后转移到PVDF膜上,用生物素标记的ss DNA与PVDF膜上的蛋白共同孵育,获得能与靶蛋白特异结合的适配体,最后通过生物素-链霉亲和素-辣根过氧化物酶系统、基因克隆测序、MEME在线软件和RNAstructure软件分析适配体的一、二级结构,并对筛选得到的适配体进行鉴定。结果:经过4轮筛选,获得了能特异识别靶蛋白而不识别无关蛋白的适配体,原库Gp45则与上述蛋白均没有结合。结论:建立了Western印迹-SELEX技术,可用于未纯化蛋白样品核酸适配体筛选。