Ultrasensitive protein-DNA binding assays

Protein-DNA binding assays have been used in a variety of fields from fundamental studies regarding the binding process itself, to serving as probes for the detection, quantification and separation of target analytes. These assays have been used for the study of protein-DNA complex stoichiometry, the detection of DNA damage, and real-time separation of free and bound complexes by electrophoretic mobility. Synthetic DNA oligonucleotides, known as aptamers, have been increasingly used for affinity binding assays to proteins, as well as for separation studies and as biosensors. Recent advances have been made in protein-DNA binding assays using capillary electrophoresis, laser-induced fluorescence, fluorescence polarization, molecular beacons, and affinity chromatography.     

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。     

High-affinity five/six-letter DNA aptamers with superior specificity enabling the detection of dengue NS1 protein variants beyond the serotype identification

Genetic alphabet expansion of DNA by introducing unnatural bases (UBs), as a fifth letter, dramatically augments the affinities of DNA aptamers that bind to target proteins. To determine whether UB-containing DNA (UB-DNA) aptamers obtained by affinity selection could spontaneously achieve high specificity, we have generated a series of UB-DNA aptamers (K_D: 27−182 pM) targeting each of four dengue non-structural protein 1 (DEN-NS1) serotypes. The specificity of each aptamer is remarkably high, and the aptamers can recognize the subtle variants of DEN-NS1 with at least 96.9% amino acid sequence identity, beyond the capability of serotype identification (69−80% sequence identities). Our UB-DNA aptamers specifically identified two major variants of dengue serotype 1 with 10-amino acid differences in the DEN-NS1 protein (352 aa) in Singaporeans’ clinical samples. These results suggest that the high-affinity UB-DNA aptamers generated by affinity selection also acquire high target specificity. Intriguingly, one of the aptamers contained two different UBs as fifth and sixth letters, which are essential for the tight binding to the target. These two types of unnatural bases with distinct physicochemical properties profoundly expand the potential of DNA aptamers. Detection methods incorporating the UB-DNA aptamers will facilitate precise diagnoses of viral infections and other diseases.     

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.     

Dimerization of an aptamer generated from Ligand-guided selection (LIGS) yields a high affinity scaffold against B-cells

Nucleic Acid Aptamers (NAAs) are a class of synthetic DNA or RNA molecules that bind specifically to their target. We recently introduced an aptamer termed R1.2 against membrane Immunoglobulin M (mIgM) expressing B-cell neoplasms using Ligand Guided Selection (LIGS). While LIGS-generated aptamers are highly specific, their lower affinity prevents aptamers from being used for translational applications. Highly specific aptamers with higher affinity can increase targetability, boosting the application of aptamers as diagnostic and therapeutic molecules. Herein, we report that dimerization of R1.2, an aptamer generated from LIGS, leads to high affinity variants without compromising the specificity. Three dimeric aptamer analogues with variable linker lengths were designed to evaluate the effect of linker length in affinity. The optimized dimeric R1.2 against cultured B-cell neoplasms, four donor B-cell samples and mIgM-positive Waldenström's Macroglobulinemia (WM) showed specificity. Furthermore, confocal imaging of dimeric aptamer and anti-IgM antibody in purified B-cells suggests co-localization. Binding assays against IgM knockout Burkitt's Lymphoma cells utilizing CRISPR/Cas9 further validated specificity of dimeric R1.2. Collectively, our findings show that LIGS-generated aptamers can be re-engineered into dimeric aptamers with high specificity and affinity, demonstrating wide-range of applicability of LIGS in developing clinically practical diagnostic and therapeutic aptamers.     

Isolation of a new ssDNA aptamer against staphylococcal enterotoxin B based on CNBr‐activated sepharose‐4B affinity chromatography

Staphylococcus aureus are potent human pathogens possessing arsenal of virulence factors. Staphylococcal food poisoning (SFP) and respiratory infections mediated by staphylococcal enterotoxin B (SEB) are common clinical manifestations. Many diagnostic techniques are based on serological detection and quantification of SEB in different food and clinical samples. Aptamers are known as new therapeutic and detection tools which are available in different ssDNA, dsDNA and protein structures. In this study, we used a new set of ssDNA aptamers against SEB. The methods used included preparation of a dsDNA library using standard SEB protein as the target analyte, affinity chromatography matrix in microfuge tubes, SELEX procedures to isolate specific ssDNA‐aptamer as an affinity ligand, aptamer purification using ethanol precipitation method, affinity binding assay using ELISA, aptamer cloning and specificity test. Among 12 readable sequences, three of them were selected as the most appropriate aptamer because of their affinity and specificity to SEB. This study presents a new set of ssDNA aptamer with favorable selectivity to SEB through 12 rounds of SELEX. Selected aptamers were used to detect SEB in infected serum samples. Results showed that SEB c1 aptamer (2 µg SEB/100 nM aptamer) had favorable specificity to SEB (k_d = 2.3 × 10^?11). In conclusion, aptamers can be considered as useful tools for detecting and evaluating SEB. The results showed that affinity chromatography was an affordable assay with acceptable accuracy to isolate sensitive and selective novel aptamers. Copyright © 2016 John Wiley & Sons, Ltd.     

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.     

Aptamer selection for the detection of Escherichia coli K88

In this study, the first group of single-stranded DNA aptamers that are highly specific to enterotoxigenic Escherichia coli (ETEC) K88 was obtained from an enriched oligonucleotide pool by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, during which the K88 fimbriae protein was used as the target and bovine serum albumin as counter targets. These aptamers were applied successfully in the detection of ETEC K88. They were then grouped under different families based on the similarity of their secondary structure and the homology of their primary sequence. Four sequences from different families were deliberately chosen for further characterization by fluorescence analysis. Having the advantage of high sensitivity, fluorescence photometry was selected as single-stranded DNA quantification method during the SELEX process. Aptamers with the highest specificity and affinity were analyzed to evaluate binding ability with E. coli. Since ETEC K88 is the only type of bacterium that expressed abundant K88 fimbriae, the selected aptamers against the K88 fimbriae protein were able to specifically identify ETEC K88 among other bacteria. This method of detecting ETEC K88 by aptamers can also be applied to bacteria other than ETEC K88.     

Application of aptamers in diagnostics,drug-delivery and imaging

Aptamers are small, single-stranded oligonucleotides (DNA or RNA) that bind to their target with high specificity and affinity. Although aptamers are analogous to antibodies for a wide range of target recognition and variety of applications, they have significant advantages over antibodies. Since aptamers have recently emerged as a class of biomolecules with an application in a wide array of fields, we need to summarize the latest developments herein. In this review we will discuss about the latest developments in using aptamers in diagnostics, drug delivery and imaging. We begin with diagnostics, discussing the application of aptamers for the detection of infective agents itself, antigens/toxins (bacteria), biomarkers (cancer), or a combination. The ease of conjugation and labelling of aptamers makes them a potential tool for diagnostics. Also, due to the reduced off-target effects of aptamers, their use as a potential drug delivery tool is emerging rapidly. Hence, we discuss their use in targeted delivery in conjugation with siRNAs, nanoparticles, liposomes, drugs and antibodies. Finally, we discuss about the conjugation strategies applicable for RNA and DNA aptamers for imaging. Their stability and self-assembly after heating makes them superior over protein-based binding molecules in terms of labelling and conjugation strategies.     

Generation of inhibitory DNA aptamers against human hepatocyte growth factor

Hepatocyte growth factor (HGF), a multifunctional cytokine, can act on many cell types. It is involved in cancer growth and metastasis by enhancing the motility of cancer cells and stimulating angiogenesis. The development of effective inhibitors for HGF is an important issue in cancer therapy. In this study, we isolated DNA aptamers against human HGF using the systematic evolution of ligands by exponential enrichment method. The selected DNA aptamers had a highly conserved consensus sequence, and could be divided into two major classes (classes I and II). The consensus motif of classes I and II might contribute to the formation of a hairpin loop structure and a G-quartet structure, respectively. These DNA aptamers bound to human HGF with high affinity and specificity. The dissociation constants of typical aptamers H38-15 and H38-21, representative of the two classes, were calculated to be approximately 20 nM. H38-15 and H38-21 inhibited the biological activities of HGF including the stimulation of scattering, migration, and invasion of pancreatic cancer KP-3 cells. Furthermore, both aptamers inhibited HGF-induced tube formation by human umbilical vein endothelial cells. These results suggested that the isolated DNA aptamers will be useful as therapeutic and diagnostic reagents for cancers.     

适配体结合蛋白质靶标的亲和力表征方法及其在疾病诊断中的应用

核酸适配体是通过体外指数富集配体系统进化（SELEX）技术筛选获得,并能够和蛋白质靶标高特异性、高亲和力结合的单链寡核苷酸。核酸适配体不但具有抗体的识别特性,而且具有自己独特的优良性能,目前已应用于分析检验、食品安全和生物医药等各个领域。蛋白质具有多种多样的生物功能以及临床诊断价值。因此,核酸适配体针对蛋白质靶标并在蛋白质相关的基础研究领域受到广泛的关注。核酸适配体应用性能的优劣取决于与其靶标蛋白质的亲和力与特异性。本文主要综述核酸适配体对蛋白质靶标的亲和力表征方法,以及在药物研发、肿瘤检测、生物成像以及生物传感器方面的应用。     

A DNA aptamer with high affinity and specificity for therapeutic anthracyclines

We describe the characterization of a DNA aptamer that displays high affinity and specificity for the anthracyclines daunomycin and doxorubicin, both of which are frequently used in chemotherapy. Aptamers were isolated from a pool of random sequences using a semiautomated procedure for magnetic beads. All selected aptamers displayed high affinity for the target molecule daunomycin. One aptamer was further characterized and exhibited a dissociation constant (KD) of 20 nM. To examine the aptamer's binding properties and clarify its applicability for diagnostic assays, its performance under various buffer conditions was evaluated. The aptamer proved to be very robust and not dependent on the presence of specific ions. It also tolerated a wide pH range and immobilization via 5'-biotinylation. Furthermore, a competition assay for sensitive daunomycin detection was established. This not only allows the determination of the aptamer's specificity but also allows the quantification of as little as 8.4 microg/L daunomycin and doxorubicin.     

ssDNA aptamers that selectively bind oxytetracycline

Single stranded DNA aptamers that bind with high affinity and specificity to the oxytetracycline (OTC) were identified by selection from an oligonucleotide library of 10(15) molecules. The binding affinities of four aptamers were in nanomolar range. The aptamers were highly selective in that, lack of -OH group at 5-position in tetracycline and -H group in place of -OH at 6-position in doxycycline determined the specificity of these aptamers to bind OTC. Three aptamers designated as No. 4, 5, and 20 shared strong affinities with K(d)=9.61, 12.08, and 56.84 nM, respectively, as well as selectivity to bind OTC (72-76%). Aptamer No. 4 had strong affinity among all with high selectivity, whereas No. 2 had relatively weak affinity (K(d)=121.1 nM) and moderate selectivity (52%). Our results indicated that the aptamers No. 4, 5, and 20 with variable 40-base oligonucleotides can be good candidates for selectively binding to OTC with high molecular discrimination over its analogs such as tetracycline and doxycycline.     

Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K_d 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.     

Chip-based detection of hepatitis C virus using RNA aptamers that specifically bind to HCV core antigen

The development of reagents with high affinity and specificity to the antigens of hepatitis C virus (HCV) is important for the early stage diagnosis of its infection. Aptamers are short, single-stranded oligonucleotides with the ability to specifically recognize target molecules with high affinity. Herein, we report the selection of RNA aptamers that bind to the core antigen of HCV. High affinity aptamers were isolated from a 10(15) random library of 60 mer RNAs using the SELEX procedure. Importantly, the selected aptamers specifically bound to the core antigen, but not to another HCV antigen, NS5, in a protein chip-based assay. Using these aptamers, we developed an aptamer-based biosensor for HCV diagnosis and detected the core antigen from HCV infected patients' sera with good specificity. This novel aptamer-based antigen detection sensor could be applied to the early diagnosis of HCV infection.     

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。     

Therapeutic aptamers: developmental potential as anticancer drugs

Aptamers, composed of single-stranded DNA or RNA oligonucleotides that interact with target molecules through a specific three-dimensional structure, are selected from pools of combinatorial oligonucleotide libraries. With their high specificity and affinity for target proteins, ease of synthesis and modification, and low immunogenicity and toxicity, aptamers are considered to be attractive molecules for development as anticancer therapeutics. Two aptamers - one targeting nucleolin and a second targeting CXCL12 - are currently undergoing clinical trials for treating cancer patients, and many more are under study. In this mini-review, we present the current clinical status of aptamers and aptamer-based cancer therapeutics. We also discuss advantages, limitations, and prospects for aptamers as cancer therapeutics. [BMB Reports 2015; 48(4): 234-237]     

Efficient isolation and elution of cellular proteins using aptamer-mediated protein precipitation assay

Protein precipitation is one of the most widely used methods for antigen detection and purification in biological research. We developed a reproducible aptamer-mediated magnetic protein precipitation method that is able to efficiently capture, purify and isolate the target proteins. We discovered DNA aptamers having individually high affinity and specificity against human epidermal growth factor receptor (EGFR) and human insulin receptor (INSR). Using aptamers and magnetic beads, we showed it is highly efficient technique to enrich endogenous proteins complex and is applicable to identify physiologically relevant protein–protein interactions with minimized nonspecific binding of proteins. The results presented here indicate that aptamers would be applicable as a useful and cost-effective tool to identify the presence of the particular target protein with their specific protein partners.     

Bi-specific aptamers mediating tumor cell lysis

Antibody-dependent cellular cytotoxicity plays a pivotal role in antibody-based tumor therapies and is based on the recruitment of natural killer cells to antibody-bound tumor cells via binding of the Fcγ receptor III (CD16). Here we describe the generation of chimeric DNA aptamers that simultaneously bind to CD16α and c-Met, a receptor that is overexpressed in many tumors. By application of the systematic evolution of ligands by exponential enrichment (SELEX) method, CD16α specific DNA aptamers were isolated that bound with high specificity and affinity (91 pm-195 nm) to their respective recombinant and cellularly expressed target proteins. Two optimized CD16α specific aptamers were coupled to each of two c-Met specific aptamers using different linkers. Bi-specific aptamers retained suitable binding properties and displayed simultaneous binding to both antigens. Moreover, they mediated cellular cytotoxicity dependent on aptamer and effector cell concentration. Displacement of a bi-specific aptamer from CD16α by competing antibody 3G8 reduced cytotoxicity and confirmed the proposed mode of action. These results represent the first gain of a tumor-effective function of two distinct oligonucleotides by linkage into a bi-specific aptamer mediating cellular cytotoxicity.