Non-SELEX selection of aptamers

Aptamers are typically selected from libraries of random DNA (or RNA) sequences by SELEX, which involves multiple rounds of alternating steps of partitioning and PCR amplification. Here we report, for the first time, non-SELEX selection of aptamers-a process that involves repetitive steps of partitioning with no amplification between them. A highly efficient affinity method, non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), was used for partitioning. We found that three steps of NECEEM-based partitioning in the non-SELEX approach were sufficient to improve the affinity of a DNA library to a target protein by more than 4 orders of magnitude. The resulting affinity was higher than that of the enriched library obtained in three rounds of NECEEM-based SELEX. Remarkably, NECEEM-based non-SELEX selection took only 1 h in contrast to several days or several weeks required for a typical SELEX procedure by conventional partitioning methods. In addition, NECEEM-based non-SELEX allowed us to accurately measure the abundance of aptamers in the library. Not only does this work introduce an extremely fast and economical method for aptamer selection, but it also suggests that aptamers may be much more abundant than they are thought to be. Finally, this work opens the opportunity for selection of drug candidates from libraries of small molecules, which cannot be PCR-amplified and thus are not approachable by SELEX.     

毛细管电泳在核酸适配体研究及核酸适配体筛选中的应用

核酸适配体是指通过指数富集配体系统进化(SELEX)技术从随机寡核苷酸文库中筛选得到的高亲和性与特异性的寡核苷酸序列配体。毛细管电泳是高效、快速、低成本的微量分离分析技术。应用毛细管电泳高效、快速筛选核酸适配体是近几年出现的新方法。本文介绍了核酸适配体筛选过程中的主要分离方法如亲和色谱、醋酸纤维素膜、凝胶电泳和磁性分离等方法,并对近年来毛细管电泳在核酸适配体中的亲和作用研究以及用于核酸适配体筛选(CE-SELEX)的主要方法(ECEEM,NECEEM,Non-SELEX和三者比较)和研究进展进行了综述。     

毛细管电泳法高效筛选8-氧代鸟嘌呤DNA糖基化酶的核酸适配体

8-氧代鸟嘌呤DNA糖基化酶(OGG1)是人体中重要的功能蛋白,在修复DNA氧化性损伤过程中起关键作用。氧化应激等引起的氧化损伤易导致炎症反应的发生,对OGG1的抑制可以一定程度上起到缓解作用;对癌细胞OGG1的抑制有望作为癌症治疗的新方法。目前的研究多集中于小分子对OGG1功能的影响和调控,而OGG1的适配体筛选尚未见报道。作为功能配体,适配体具有合成简单、高亲和力及高特异性等优点。该文筛选了OGG1的核酸适配体,结合毛细管电泳高效快速的优点建立了两种基于毛细管电泳-指数富集进化(CE-SELEX)技术的筛选方法:同步竞争法和多轮筛选法。同步竞争法利用单链结合蛋白(SSB)与核酸库中单链核酸的强结合能力,与目标蛋白OGG1组成竞争体系,并通过增加SSB浓度来增加竞争筛选压力,以去除与OGG1弱结合的核酸序列,一步筛选即可获得与OGG1强结合的核酸序列。多轮筛选法在相同孵育条件和电泳条件下,经3轮筛选获得OGG1的核酸适配体。比较两种筛选方法的筛选结果,筛选结果中频次最高的3条候选核酸适配体序列一致,其解离常数(K_D)值在1.71~2.64 μmol/L之间。分子对接分析结果表明候选适配体1(Apt 1)可能与OGG1中具有修复氧化性损伤功能的活性口袋结合。通过对两种筛选方法的对比,证明同步竞争法更加快速高效,对其他蛋白核酸适配体筛选方法的选择具有一定的指导意义。得到的适配体有望用于OGG1功能调控,以抑制其修复功能。     

蛋白质的核酸适配体筛选的研究进展

核酸适配体是通过指数富集系统配体进化(SELEX)筛选获得的,与靶标具有高亲和力和特异性结合的单链DNA或RNA。蛋白质是生命进程中的关键功能分子。近年来,以蛋白质为靶标的适配体筛选在蛋白质相关的基础及应用研究领域受到广泛关注。核酸适配体应用性能的优劣取决于其亲和力、特异性与稳定性。目前,适配体筛选方法的优化主要是提高筛选效率、提升适配体性能及降低筛选成本。适配体主要筛选步骤包括复合物分离、核酸库优化、次级库的富集、适配体序列分析以及亲和力表征等。迄今为止,以蛋白质-核酸复合物的分离为核心步骤的适配体筛选方法有20余种。本文归纳总结了2005年以来以蛋白质为靶标的适配体筛选技术,讨论了各方法的缺陷与局限。介绍了核酸库的设计优化方法、适配体的序列特征,以及常用的亲和力表征方法。     

Selection of aptamers for signal transduction proteins by capillary electrophoresis

High‐affinity aptamers for important signal transduction proteins, i.e. Cdc42‐GTP, p21‐activated kinase1 (PAK1) and MRCK (myotonic dystrophy kinase‐related Cdc42‐binding kinase) α were successfully selected in the low micro‐ to nanomolar range using non‐systematic evolution of ligands by exponential enrichment (SELEX) with at least three orders of magnitude enhancement from their respective bulk affinity of naïve DNA library. In the non‐SELEX procedure, CE was used as a highly efficient affinity method to select aptamers for the desired molecular target through a process that involved repetitive steps of partitioning, known as non‐equilibrium CE of equilibrium mixtures with no PCR amplification between successive steps. Various non‐SELEX conditions including the type, concentration and pH of the run buffer were optimized. Other considerations such as salt composition of selection buffer, protein concentration and sample injection size were also studied for high stringency during selection. After identifying the best enriched aptamer pool, randomly selected clones from the aptamer pool were sequenced to obtain the individual DNA sequences. The dissociation constants (K_d) of these sequences were in the low micromolar to nanomolar range, indicating high affinity to the respective proteins. The best binders were also subjected to sequence alignment to generate a phylogenetic tree. No significant consensus region based on approximately 50 sequences for each protein was observed, suggesting the high efficiency of non‐SELEX for the selection of numerous unique sequences with high selectivity.     

Methods developed for SELEX

SELEX (systematic evolution of ligands by exponential enrichment) is a process that involves the progressive purification from a combinatorial library of nucleic acid ligands with a high affinity for a particular target by repeated rounds of partitioning and amplification. With the development of aptamer technology over the last decade, various modified SELEX processes have arisen that allow various aptamers to be developed against a wide variety of molecules, irrespective of the target size. In the present review, the separation methods used in such SELEX processes are reviewed.     

Capillary electrophoresis as a tool for screening aptamer with high affinity and high specificity to ricin

Aptamers which specifically recognize targets are selected from random oligonucleotide library using systematic evolution of ligands by exponential enrichment (SELEX). In this paper, capillary electrophoresis (CE) as a separation approach has been introduced to SELEX procedure. The high efficiency of CE gives rise to greatly shorten the selection procedure. The results from enzyme-linked assay and dot blot experiment show that an enrichment pool has been obtained after four rounds selection, which can specifically recognize ricin. __________ Translated from Chemical Journal of Chinese Universities, 2006, 27(10): 1,840–1,843 [译自: 高等学校化学学报]     

Kinetic capillary electrophoresis-based affinity screening of aptamer clones

DNA aptamers are single stranded DNA (ssDNA) molecules artificially selected from random-sequence DNA libraries for their specific binding to a certain target. DNA aptamers have a number of advantages over antibodies and promise to replace them in both diagnostic and therapeutic applications. The development of DNA aptamers involves three major stages: library enrichment, obtaining individual DNA clones, and the affinity screening of the clones. The purpose of the screening is to obtain the nucleotide sequences of aptamers and the binding parameters of their interaction with the target. Highly efficient approaches have been recently developed for the first two stages, while the third stage remained the rate-limiting one. Here, we introduce a new method for affinity screening of individual DNA aptamer clones. The proposed method amalgamates: (i) aptamer amplification by asymmetric PCR (PCR with a primer ratio different from unity), (ii) analysis of aptamer-target interaction, combining in-capillary mixing of reactants by transverse diffusion of laminar flow profiles (TDLFP) and affinity analysis using kinetic capillary electrophoresis (KCE), and (iii) sequencing of only aptamers with satisfying binding parameters. For the first time we showed that aptamer clones can be directly used in TDLFP/KCE-based affinity analysis without an additional purification step after asymmetric PCR amplification. We also demonstrated that mathematical modeling of TDLFP-based mixing allows for the determination of K_d values for the in-capillary reaction of an aptamer and a target and that the obtained K_d values can be used for the accurate affinity ranking of aptamers. The proposed method does not require the knowledge of aptamer sequences before screening, avoids lengthy (3-5 h) purification steps of aptamer clones, and minimizes reagent consumption to nanoliters.     

Selection of DNA Aptamers with Affinity for Pro-gastrin-Releasing Peptide (proGRP), a Tumor Marker for Small Cell Lung Cancer

Aptamers are single-strand oligonucleotides that are generated by the systemic evolution of ligands by exponential enrichment (SELEX) technique and that can bind to target molecules specifically. However, only a few aptamers have been developed to date against tumor markers. To utilize aptamers for tumor diagnosis, a variety of aptamers are required. Here, a single-stranded DNA aptamer specific for pro-gastrin-releasing peptide (proGRP), a marker for small cell lung cancer, was selected using SELEX. After selection, identical sequences were found in the DNA library. This sequence was selected and its binding affinity to proGRP was evaluated using surface plasmon resonance.     

细菌的核酸适配体筛选的研究进展

核酸适配体(aptamer)是通过指数富集配体系统进化技术(SELEX)筛选的能够以高亲和力和高特异性识别靶标分子或细胞的核糖核酸(RNA)和单链脱氧核糖核酸(ssDNA)。作为化学抗体,核酸适配体的制备和合成比抗体的成本更低。核酸适配体的靶标范围极其广泛,包括小分子、生物大分子、细菌和细胞等。针对细菌靶标筛选的适配体,目前主要应用于食品、医药和环境中的细菌检测。细菌的核酸适配体筛选可以通过离心法将菌体-适配体复合物与游离的适配体分离,并通过荧光成像、荧光光谱分析、流式细胞仪分选、DNA捕获元件、酶联适配体分析等方法表征适配体与靶标的相互作用。筛选出的适配体可结合生物、化学检测方法用于细菌检测。本文介绍了细菌适配体的筛选和表征方法以及基于适配体的检测方法的最新进展,分析了不同检测方法的利弊,并列出了2011~2015年筛选的细菌的核酸适配体。     

复合靶指数富集的配基系统进化技术研究进展

核酸适配体是一类具有高度特异性和亲和力的单链寡核苷酸,被誉为"人工单抗",具有广阔的应用前景。它一般是通过指数富集的配基系统进化（SELEX）技术筛选获得。目前SELEX技术多局限于单一、纯化的可溶性蛋白质靶标。然而,蛋白质的纯化过程繁琐,耗时费力,而且很多靶标（如血清中的低丰度蛋白质或细胞的膜蛋白）很难纯化获得单一纯品。复合靶SELEX技术则可以避免靶标的纯化过程,能够保持靶标的天然构象,并且可以在未明确靶标的组成及结构特性的前提下,通过高通量的盲筛获得一系列特异性核酸适配体。该文主要介绍以未纯化的各种生物样本为复合靶的SELEX技术,以期为核酸适配体的筛选提供新思路。     

New trends in affinity sensing: aptamers for ligand binding

Aptamers are artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules. They are isolated from complex libraries of synthetic nucleic acids by an iterative process of adsorption, recovery and amplification. This review described the in vitro process to obtain aptamers (SELEX). It mentions the main characteristics of these molecules (i.e. affinity, specificity and stability). Moreover, it discusses advantages over antibodies. It reports potential applications of aptamers in analytical and diagnostic assays as biocomponents of biosensors (aptasensors) and allosteric ribozymes (aptazymes).     

全细胞的核酸适配体筛选的研究进展

核酸适配体(aptamer)是从人工合成的随机单链DNA(ssDNA)或RNA文库中筛选得到的,能够高亲和力、高特异性地与靶标结合的ssDNA或RNA。核酸适配体的靶标范围广,可包括小分子、蛋白质、细胞、微生物等多种靶标。其中以细胞为靶标的适配体在生物感应、分子成像、医学诊断、药物传输和疾病治疗等领域有很大的应用潜能。但全细胞的核酸适配体筛选过程复杂,筛选难度大,筛选的适配体性能不佳是导致目前可用的适配体非常有限的主要原因。由于细胞表面蛋白质在提取纯化过程中分子结构和形态会发生改变,故以膜表面蛋白质为靶标筛选的适配体很难应用于识别整体细胞。以全细胞为靶标的核酸适配体筛选则不需要准确了解细胞表面的分子结构,筛选过程中可保持细胞的天然状态,以全细胞为靶标筛选出的核酸适配体有望直接用于全细胞识别。本文总结了2008~2015年全细胞的核酸适配体筛选的研究进展,介绍了靶细胞的分类、核酸库的设计、筛选条件和方法以及核酸适配体的亲和力表征方法等。并列出全细胞靶标的核酸适配体序列。     

Electrochemical nanomaterial-based nucleic acid aptasensors

Recent progress in the development of electrochemical nanomaterial–aptamer-based biosensors is summarized. Aptamers are nucleic acid ligands that can be generated against amino acids, drugs, proteins, and other molecules. They are isolated from a large random library of synthetic nucleic acids by an iterative process of binding, separation, and amplification, called systematic evolution of ligands by exponential enrichment (SELEX). In this review, different methods of integrating aptamers with different nanomaterials and nanoparticles for electrochemical biosensing application are described.     

采用毛细管电泳技术筛选特异识别蓖麻毒素适配子的研究

采用指数富集配基的系统进化(SELEX)技术从随机寡核苷酸文库中筛选获得特异识别蓖麻毒素靶分子的适配子. 将毛细管电泳技术作为分离手段引入到SELEX筛选中, 利用毛细管电泳高效的分离能力使得筛选周期大大缩短. 酶联免疫和斑点杂交实验结果表明, 仅经4轮筛选即可获得特异识别蓖麻毒素蛋白的寡核苷酸适配子.     

Single-stranded DNA-binding protein facilitates gel-free analysis of polymerase chain reaction products in capillary electrophoresis

It has been recently demonstrated that single-stranded DNA-binding protein (SSB) can facilitate quantitative analyses of DNA, RNA, and proteins in gel-free capillary electrophoresis (CE). Here, we report the application of SSB-mediated gel-free CE for analyses of polymerase chain reaction (PCR) products. The unique ability of SSB to bind ssDNA but not double-stranded DNA (dsDNA) allows efficient separation of three types of DNA molecules in the PCR reaction mixture: primers, products (amplified templates), and by-products, which originate from non-specific DNA hybridization. SSB-mediated gel-free CE analysis of PCR products combines simplicity, high sensitivity, and outstanding quantitative capabilities. The ability of the method to distinguish between products and by-products makes this method an indispensable tool in preparative PCR (e.g., in the development of nucleotide aptamers).     

Nonequilibrium capillary electrophoresis of equilibrium mixtures: a universal tool for development of aptamers

Aptamers are DNA (or RNA) ligands selected from large libraries of random DNA sequences and capable of binding different classes of targets with high affinity and selectivity. Both the chances for the aptamer to be selected and the quality of the selected aptamer are largely dependent on the method of selection. Here we introduce selection of aptamers by nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). The new method has a number of advantages over conventional approaches. First, NECEEM-based selection has exceptionally high efficiency, which allows aptamer development with fewer rounds of selection. Second, NECEEM can be equally used for selecting aptamers and finding their binding parameters. Finally, due to its comprehensive kinetic capabilities, the new method can potentially facilitate selection of aptamers with predefined K(d), k(off), and k(on) of the aptamer-target interaction. In this proof-of-principle work, we describe the theoretical bases of the method and demonstrate its application to a one-step selection of DNA aptamers with nanomolar affinity for protein farnesyltransferase.     

In Vitro Selection of Circular DNA Aptamers for Biosensing Applications

We report on the first effort to select DNA aptamers from a circular DNA library, which resulted in the discovery of two high‐affinity circular DNA aptamers that recognize the glutamate dehydrogenase (GDH) from Clostridium difficile, an established antigen for diagnosing Clostridium difficile infection (CDI). One aptamer binds effectively in both the circular and linear forms, the other is functional only in the circular configuration. Interestingly, these two aptamers recognize different epitopes on GDH, demonstrating the advantage of selecting aptamers from circular DNA libraries. A sensitive diagnostic test was developed to take advantage of the high stability of circular DNA aptamers in biological samples and their compatibility with rolling circle amplification. This test is capable of identifying patients with active CDI using stool samples. This work represents a significant step forward towards demonstrating the practical utility of DNA aptamers in clinical diagnosis.     

化学修饰提高核酸适体结合亲和力的研究进展

核酸适体(Aptamer)是通过体外筛选得到的短单链DNA或RNA寡核苷酸, 具有与抗体相当或更优异的特异性及亲和力, 且具有靶标范围广、 易制备和灵活可控修饰、 免疫原性低、 批次差异性小以及易于运输保存等优势, 为食品、 环境和生物医学等领域提供了全新的分子识别工具, 获得了研究者的广泛关注. 但是目前其商业应用的数量仍有限. 为了增强核酸适体的应用性能, 研究者对核酸适体进行了大量的改性研究. 本文系统总结了核酸适体筛选前、 后采用非共价或共价方式对其进行化学修饰, 以增加核酸适体与靶标的结合亲和力的相关研究进展, 并对未来发展前景进行了展望.     

A Mathematical Analysis of the Selective Enrichment of NECEEM-Based Non-SELEX

Non-Systematic Evolution of Ligands by EXponential enrichment (SELEX)selection of aptamers, a novel technology for aptamer selection from libraries of random DNA (or RNA) sequences, involves repetitive steps of partitioning without polymerase chain reaction (PCR) amplification between them. This selection is based on non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) and has exceptionally high efficiency. In this paper, a mathematical analysis was carried out to predict the levels of enrichment of non-SELEX selection under different conditions such as different protein concentrations and different efficiencies of partitioning. Investigated results suggest that the magnitude of the bulk affinity (k _d) being 10⁴ or 10⁵ μM for the initial pool has no obvious effect on selective enrichment and that the first, second, and third rounds of non-SELEX selection have different optimum protein concentration values [T _f] that give maximum enrichment levels when [T _f] ranges from 0.0005 to 0.5 μM. The significance of analyzing selective enrichment of NECEEM-based non-SELEX with the efficiency of partitioning target-bound ligands from free ligands has been demonstrated.