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。     

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。     

与草甘膦特异结合的核酸适配体筛选及应用

通过体外指数富集配体系统进化(SELEX)技术,筛选靶向草甘膦核酸适配体A08。使用酶联寡核苷酸测定法(ELONA)和斑点印迹确认草甘膦核酸适配体A08与草甘膦的特异性,未观察到非特异性。基于ELONA平台,草甘膦检测限为4 ng/μL。圆二色谱(CD)实验表明,草甘膦核酸适配体A08形成茎环和分子内G-四链体,可以稳定存在于结合的磷酸盐缓冲溶液中。此外,亲和力实验显示草甘膦与核酸适配体之间具有强的结合力,解离常数(K_d)为38.38±9.094 nmol/L。基于草甘膦核酸适配体A08的ELONA法测定的准确性在真正的草甘膦样品中得到证实。获得的草甘膦核酸适配体A08为制备检测草甘膦试剂盒奠定了坚实的基础。     

核酸适配体传感器在黄曲霉毒素B1检测中应用研究进展北大核心CSCD

核酸适配体作为一种新型识别分子,具有亲和力高、稳定性强、制备成本低、特异性强等优点,但其自身不具有信号转换功能,它与靶标分子特异性结合过程,不可产生被检测的物理化学信号。因此,需将核酸适配体与靶标分子特异性识别结合过程转为易于被检测的物理化学信号变化的过程。根据信号转换方式的不同,可将适配体生物传感器分为荧光适配体传感器、比色适配体传感器、电化学适配体传感器和表面拉曼散射适配体传感器。本文对基于以上4种检测信号的核酸适配体生物传感器在黄曲霉毒素(AFB1)检测方面的应用进行综述,并概述该类传感器应用前景和当前面临的挑战。     

小分子靶标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(SELEX)技术筛选得到的核糖核酸(RNA)或单链脱氧核糖核酸(ssDNA)。核酸适配体通过高亲和力特异性地识别小分子、蛋白质、细胞、微生物等多种靶标,在生物、医药、食品和环境检测等领域的应用日渐增多。但目前实际可用的核酸适配体有限,其筛选过程复杂,筛选难度大,制约了其应用。与生物大分子、细胞和微生物等靶标不同,小分子靶标与核酸分子的结合位点少、亲和力弱,且靶标通常需要固定在载体上。此外,小分子靶标结合核酸形成的复合物与核酸自身的大小、质量、电荷性质等方面差异较小,二者的分离难度大。故小分子靶标的核酸适配体筛选过程与大分子和细胞等复合靶标相比有明显差异,筛选难度更大。因此需要根据其自身结构特点和核酸适配体的应用目的选定靶标或核酸库的固定方法,优化靶标核酸复合物的分离方法。本文介绍了不同类型小分子(具有基团差异的单分子、含相同基团分子和手性分子等)靶标的选择及其核酸适配体的筛选方法,并对核酸库的设计、与靶标结合的核酸的分离方法和亲和作用表征方法进行了介绍,列出了自2008年以来报道的40余种小分子靶标的核酸适配体序列和复合物的平衡解离常数(K_d)。     

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。     

核酸适配体荧光探针在生化分析和生物成像中的研究进展

核酸适配体是指通过体外筛选技术从核酸文库中筛选出来,能够高特异性、高亲和力识别靶标物的寡核苷酸序列,具有靶标类型广泛、合成简单、相对分子质量小、化学稳定性高、易于进行生物化学修饰等优点。 核酸适配体能够通过折叠成特定的二维或三维构型与靶标物特异性结合,加上合适的信号转导机制,为重要靶标物的研究提供理想的分子识别与分子检测探针。 荧光检测技术具有高灵敏、高分辨率、易于实现多元分析等优点。 将核酸适配体的分子识别特性与荧光优异的光学检测性能相结合,在生命科学研究领域有着广泛的应用空间。 本文主要综述了核酸适配体荧光探针常见的分子设计和信号响应方式,及其在细胞成像、亚细胞成像中的应用研究,并对核酸适配体探针目前面临的一些挑战进行了讨论,最后对其未来的发展方向进行了展望。     

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。     

基于核酸适配体和纳米粒子的光学探针

核酸适配体(aptamer)是一类通过指数富集的配体系统进化技术(SELEX)经体外筛选得到的单链DNA或RNA。核酸适配体借自身形成的空间结构与靶标分子特异性结合,具有靶分子广、亲和力高、特异性强、易改造修饰等特点,因而在生命科学、临床诊断、药物发现和环境科学等方面得以广泛应用。近年来,核酸适配体与纳米技术结合,并利用纳米材料在光学、磁学、电学、化学及生物学方面表现出的特殊性质,实现了对靶标分子高灵敏度、高选择性、简便快速的识别与检测。本文评述了基于核酸适配体-纳米粒子特性的光学探针在生物大分子、金属离子和有机小分子检测等领域的应用现状与发展趋势,主要包括比色法、荧光光谱法、表面增强拉曼光谱法等。     

前列腺癌细胞核酸适配体支持向量机分类模型

本文将前列腺癌(Prostate Cancer,PCa)PC-3M-1E8细胞为标靶的核酸适配体序列翻译成氨基酸序列,计算氨基酸序列的分子参数,然后用这些分子参数建立核酸适配体亲和性的构-效关系模型。所用的候选核酸适配体序列是采用以细胞为靶标的指数富集配体系统进化(Cell-SELEX)技术筛选得到。模型训练集、测试集分别包含150、50条核酸序列,均由第3轮和11轮的候选核酸适配体组成。将第3轮的核酸序列类标签值设置为"1",代表低亲和性、低特异性的候选核酸适配序列;将第11轮筛选所得核酸序列类标签值设置为"2",代表高亲和性、高特异性候选核酸适配序列。基于二值分类问题的支持向量机分类(SVC)算法用于建模。SVC模型对训练集、测试集的预测准确度分别为87.3%、86%。另外,采用SVC模型对第5、7、9轮的序列也进行了预测。第3、5、7、9、11轮的高亲和性与高特异性核酸适配体的分率分别是0.23、0.41、0.61、0.64、0.87,预测结果符合SELEX筛选的适配体进行规律。     

Screening and preliminary application of a DNA aptamer for rapid detection of Salmonella O8

We report on a rapid method for the detection of Salmonella O8. It does not require an enrichment step but rather uses an aptamer as a probe that was selected by system evolution of ligands by exponential enrichment (SELEX) assay. Firstly, aptamer against Salmonella O8 was selected from a 78 bp random DNA library that was prepared in-vitro. The binding ability of the aptamers to target bacterium was examined by aptamer-linked immobilized sorbent assay. A high affinity aptamer was successfully selected from the initial random DNA pool, and its secondary structure was also investigated. Next, this high affinity aptamer B10 was used to recognize Salmonella O8 via fluorescence microscopy. The selected aptamer has a high specificity and high affinity against its target. We believe that the resulting fluorescence in-situ labeling assay is a potentially useful alternative in rapid screening and detection of foodborne pathogens.

Highly Specific Recognition of Guanosine Using Engineered Base-Excised Aptamers

Purines and their derivatives are highly important molecules in biology for nucleic acid synthesis, energy storage, and signaling. Although many DNA aptamers have been obtained for binding adenine derivatives such as adenosine, adenosine monophosphate, and adenosine triphosphate, success for the specific binding of guanosine has been limited. Instead of performing new aptamer selections, we report herein a base-excision strategy to engineer existing aptamers to bind guanosine. Both a Na⁺-binding aptamer and the classical adenosine aptamer have been manipulated as base-excising scaffolds. A total of seven guanosine aptamers were designed, of which the G16-deleted Na⁺ aptamer showed the highest bindng specificity and affinity for guanosine with an apparent dissociation constant of 0.78 mm . Single monophosphate difference in the target molecule was also recognizable. The generality of both the aptamer scaffold and excised site were systematically studied. Overall, this work provides a few guanosine binding aptamers by using a non-SELEX method. It also provides deeper insights into the engineering of aptamers for molecular recognition.     

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.     

Probing high-affinity 11-mer DNA aptamer against Lup an 1 (β-conglutin)

Aptamers are synthetic nucleic acids with great potential as analytical tools. However, the length of selected aptamers (typically 60–100 bases) can affect affinity, due to the presence of bases not required for interaction with the target, and therefore, the truncation of these selected sequences and identification of binding domains is a critical step to produce potent aptamers with higher affinities and specificities and lowered production costs. In this paper we report the truncation of an aptamer that specifically binds to β-conglutin (Lup an 1), an anaphylactic allergen. Through comparing the predicted secondary structures of the aptamers, a hairpin structure with a G-rich loop was determined to be the binding motif. The highest affinity was observed with a truncation resulting in an 11-mer sequence that had an apparent equilibrium dissociation constant (K _D) of 1.7?×?10^?9 M. This 11-mer sequence was demonstrated to have high specificity for β-conglutin and showed no cross-reactivity to other lupin conglutins (α-, δ-, γ-conglutins) and closely related proteins such as gliadin. Finally, the structure of the truncated 11-mer aptamer was preliminarily elucidated, and the GQRS Mapper strongly predicted the presence of a G-quadruplex, which was subsequently corroborated using one-dimensional NMR, thus highlighting the stability of the truncated structure.     

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。     

Cocaine detection by structure-switch aptamer-based capillary zone electrophoresis

Aptamers, which are nucleic acid oligonucleotides that can bind targets with high affinity and specificity, have been widely applied as affinity probes in capillary electrophoresis (CE). Due to relative weak interaction between aptamers and small molecules, the application of aptamer-based CE is still limited in certain compounds. A new strategy that is based on the aptamer structure-switch concept was designed for small molecule detection by a novel CE method. A carboxyfluorescein (fluorescein amidite, FAM) label DNA aptamer was first incubated with partial complementary strand (CS), and then the free aptamer and the aptamer-CS duplex were well separated and determined by metal cation mediated CE/laser-induced fluorescence. When the target was introduced into the incubated sample, the hybridized form was destabilized, resulting in the changes of the fluorescence intensities of the free aptamer and the aptamer-CS duplex. The length of CS was investigated and 12 mer CS showed the best sensitivity for the detection of cocaine. The presented CE-LIF method, which combines the separation power of CE with the specificity of interactions occurring between target, aptamer, and CS, could be a universal detection strategy for other aptamer-specified small molecules.     

RNA aptamers that bind flavin and nicotinamide redox cofactors

RNA molecules that specifically bind riboflavin (Rb) and beta-nicotinamide mononucleotide (NMN) have been isolated by in vitro selection. A simple structural motif containing intramolecular G-quartets was found to bind tightly to oxidized riboflavin (Kd = 1-5 micromolar). DNA versions of the consensus sequence also bind, but with weaker affinity. DMS protection experiments show that the quartet structure of these aptamers is stabilized by interaction with the flavin. As a measure of their redox specificity, the aptamers do not show significant differential binding between oxidized and reduced forms of a 5-deazariboflavin derivative that is a close structural analog of riboflavin. In contrast to the lack of redox specificity of the riboflavin aptamers, RNAs selected for binding to the nicotinamide portion of NAD discriminate between NAD and NADH in solution by over an order of magnitude. A mutagenized pool based on one of the NMN aptamer sequences was used to reselect for NMN binding. Comparison of the reselected sequences led to the identification of the binding region of the aptamer. A complex secondary structure containing two interacting stem-loops is proposed for the minimal NMN-binding RNA. The same mutagenized pool was used to select for increased discrimination between NMN and NMNH. From these reselected sequences, a mutation within the binding region was identified that increases specificity for NMN. These experiments show that RNA can bind these cofactors with low micromolar affinity and, in the case of nicotinamide cofactors, can discriminate between the two redox states. These cofactor binding motifs may provide a framework for generating new ribozymes that catalyze redox reactions similar to those found in basic metabolic pathways.     

Aptamer binding assays for proteins: The thrombin example—A review

Experimentally selected single-stranded DNA and RNA aptamers are able to bind to specific target molecules with high affinity and specificity. Many analytical methods make use of affinity binding between the specific targets and their aptamers. In the development of these methods, thrombin is the most frequently used target molecule to demonstrate the proof-of-principle. This paper critically reviews more than one hundred assays that are based on aptamer binding to thrombin. This review focuses on homogeneous binding assays, electrochemical aptasensors, and affinity separation techniques. The emphasis of this review is placed on understanding the principles and unique features of the assays. The principles of most assays for thrombin are applicable to the determination of other molecular targets.