共查询到20条相似文献,搜索用时 15 毫秒
1.
Takemura K Wang P Vorberg I Surewicz W Priola SA Kanthasamy A Pottathil R Chen SG Sreevatsan S 《Experimental biology and medicine (Maywood, N.J.)》2006,231(2):204-214
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. 相似文献
2.
High affinity target-binding aptamers are identified from random oligonucleotide libraries by an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). Since the SELEX process includes a PCR amplification step the randomized region of the oligonucleotide libraries need to be flanked by two fixed primer binding sequences. These primer binding sites are often difficult to truncate because they may be necessary to maintain the structure of the aptamer or may even be part of the target binding motif. We designed a novel type of RNA library that carries fixed sequences which constrain the oligonucleotides into a partly double-stranded structure, thereby minimizing the risk that the primer binding sequences become part of the target-binding motif. Moreover, the specific design of the library including the use of tandem RNA Polymerase promoters allows the selection of oligonucleotides without any primer binding sequences. The library was used to select aptamers to the mirror-image peptide of ghrelin. Ghrelin is a potent stimulator of growth-hormone release and food intake. After selection, the identified aptamer sequences were directly synthesized in their mirror-image configuration. The final 44 nt-Spiegelmer, named NOX-B11-3, blocks ghrelin action in a cell culture assay displaying an IC50 of 4.5 nM at 37°C. 相似文献
3.
Daniel M. Dupont Niels Larsen Jan. K. Jensen Peter A. Andreasen J?rgen Kjems 《Nucleic acids research》2015,43(21):e139
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. 相似文献
4.
5.
6.
The entrance of influenza virus into host cells is facilitated by the attachment of the globular region of viral hemagglutinin
to the sialic acid receptors on host cell surfaces. In this study, we have cloned the cDNA fragment encoding the entire globular
region (residues 101–257) of hemagglutinin of the H9N2 type avian influenza virus (A/ck/Korea/ms96/96). The protein segment
(denoted as the H9 peptide), which was expressed and purified in E. coli, was used for the immunization of BALB/c mice to obtain the anti-H9 antiserum. To identify specific DNA aptamers with high
affinity to H9 peptide, we conducted the SELEX method; 19 aptamers were newly isolated. A random mixture of these aptamers
showed an increased level of binding affinity to the H9 peptide. The sequence alignment analysis of these aptamers revealed
that 6 aptamers have highly conserved consensus sequences. Among these, aptamer C7 showed the highest similarity to the consensus
sequences. Therefore, based on the C7 aptamer, we synthesized a new modified aptamer designated as C7-35M. This new aptamer
showed strong binding capability to the viral particles. Furthermore, it could prevent MDCK cells from viral infection by
strong binding to the viral particles. These results suggest that our aptamers can recognize the hemagglutinin protein of
avian influenza virus and inhibit the binding of the virus to target receptors required for the penetration of host cells. 相似文献
7.
胃癌是发病率及死亡率均较高的消化道恶性肿瘤之一,严重威胁人类的生命健康。血清肿瘤标志物的检测对提高早期胃癌的检出率,改善胃癌的治疗有重要的意义。核酸适配体以其灵敏度高、靶向性强等优势显示出了较强的临床适用性。本研究以双向热循环消减指数富集式配基系统进化(systematic evolution of ligands by exponential enrichment,SELEX)技术为支持,纳米(琼脂)磁珠材料为载体,胃癌血清及正常人血清为筛选靶标,结合高通量测序技术建立了快速高效的核酸适配体筛选方法。经过19轮双向筛选,获得高重复率的胃癌血清特异性核酸适配体序列10条及正常人血清特异性核酸适配体序列8条。将这些序列分别混合并制成检测试剂A、B,结合实时荧光定量PCR(quantitative real-time PCR,qPCR)技术,对100份临床血清样本进行特异性验证。通过比较分析,建立了快速高效的胃癌血清检测技术。结果显示,核酸适配体G AP1与N AP1的二级结构类似于抗体的“Y”型,且呈茎环状。检测试剂A、B对胃癌及正常人血清的检出率分别为92%和88%。表明本技术可以较准确地筛选得到高特异性和强亲和力的核酸适配体,体现了核酸适配体作为新型肿瘤标志物在临床检测及治疗的应用潜力。 相似文献
8.
9.
Prostatic acid phosphatase (PAP) expression increases proportionally with prostate cancer progression, making it useful in prognosticating intermediate to high-risk prostate cancers. A novel ligand that can specifically bind to PAP would be very helpful for guiding prostate cancer therapy. RNA aptamers bind to target molecules with high specificity and have key advantages such as low immunogenicity and easy synthesis. Here, human PAP-specific aptamers were screened from a 2′-fluoropyrimidine (FY)-modified RNA library by SELEX. The candidate aptamer families were identified within six rounds followed by analysis of their sequences and PAP-specific binding. A gel shift assay was used to identify PAP binding aptamers and the 6N aptamer specifically bound to PAP with a Kd value of 118 nM. RT-PCR and fluorescence labeling analyses revealed that the 6N aptamer bound to PAP-positive mammalian cells, such as PC-3 and LNCaP. IMR-90 negative control cells did not bind the 6N aptamer. Systematic minimization analyses revealed that 50 nucleotide sequences and their two hairpin structures in the 6N 2′-FY RNA aptamer were equally important for PAP binding. Renewed interest in PAP combined with the versatility of RNA aptamers, including conjugation of anti-cancer drugs and nano-imaging probes, could open up a new route for early theragnosis of prostate cancer. 相似文献
10.
Moreno M Rincón E Piñeiro D Fernández G Domingo A Jiménez-Ruíz A Salinas M González VM 《Biochemical and biophysical research communications》2003,308(2):214-218
SELEX procedure is a methodology in which single stranded oligonucleotides are selected from a wide variety of sequences based on their interaction with a target molecule. We have designed a novel SELEX methodology using colloidal gold to select high affinity single stranded DNA aptamers against Leishmania infantum KMP-11. Kinetoplastid membrane protein-11 (KMP-11) is a major component of the cell membrane of kinetoplastid parasites. Although its function is not known, the fact that KMP-11 is a cytoskeleton-associated protein suggests that it may be involved in mobility or in some other aspects of the flagellar structure. We have isolated a single stranded DNA aptamer population that binds specifically to L. infantum KMP-11. This population has been characterized in a series of in vitro experiments suggesting that it may be used as a powerful tool to further investigate the role of KMP-11 during Leishmania development and/or as a diagnostic tool in Leishmania infection. 相似文献
11.
12.
Assays for cytokines using aptamers 总被引:2,自引:0,他引:2
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. 相似文献
13.
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(KD,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KD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。 相似文献
14.
Systematic evolution of a DNA aptamer binding to rat brain tumor microvessels. selective targeting of endothelial regulatory protein pigpen 总被引:11,自引:0,他引:11
Blank M Weinschenk T Priemer M Schluesener H 《The Journal of biological chemistry》2001,276(19):16464-16468
Tumor microvessels differ in structure and metabolic function from normal vasculature, and neoangiogenesis is associated with quantitative and qualitative changes in expression of endothelial proteins. Such molecules could serve as molecular addresses differentiating the tumor vasculature from those of the normal brain. We have applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) against transformed endothelial cells as a complex target to select single-stranded DNA-ligands (aptamers) that function as histological markers to detect microvessels of rat experimental glioma, a fatal brain tumor that is highly vascularized. Both the SELEX selection procedure as well as subsequent deconvolution-SELEX were analyzed by fluorescence based methods (flow cytometry and fluorescence microscopy). Of 25 aptamers analyzed, one aptamer was selected that selectively bound microvessels of rat brain glioblastoma but not the vasculature of the normal rat brain including peritumoral areas. The molecular target protein of aptamer III.1 was isolated from endothelial cells by ligand-mediated magnetic DNA affinity purification. This protein was identified by mass spectrometry as rat homologue of mouse pigpen, a not widely known endothelial protein the expression of which parallels the transition from quiescent to angiogenic phenotypes in vitro. Because neoangiogenesis, the formation of new blood vessels, is a key feature of tumor development, the presented aptamer can be used as a probe to analyze pathological angiogenesis of glioblastoma. The presented data show that pigpen is highly expressed in tumor microvessels of experimental rat brain glioblastoma and may play an important role in warranting blood supply, thus growth of brain tumors. 相似文献
15.
Isolation and characterization of RNA aptamers specific for the hepatitis C virus nonstructural protein 3 protease. 总被引:11,自引:0,他引:11
K Fukuda D Vishnuvardhan S Sekiya J Hwang N Kakiuchi K Taira K Shimotohno P K Kumar S Nishikawa 《European journal of biochemistry》2000,267(12):3685-3694
Nonstructural protein 3 (NS3) from hepatitis C virus (HCV) is a serine protease that provides an essential function in maturation of the virus by cleaving the nonstructural regions of the viral polyprotein. The goal of this work was to isolate RNA aptamers that bind specifically to the NS3 protease active site in the truncated polypeptide DeltaNS3. RNA aptamers were selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX). The RNA pool for SELEX had a 30-nucleotide randomized core region. After nine selection cycles, a pool of DeltaNS3-specific RNA aptamers were obtained. This RNA pool included 45 clones that divided into three main classes (G9-I, II and III). These classes include the conserved sequence GA(A/U)UGGGAC. These aptamers bind to DeltaNS3 with a binding constant of about 10 nM and inhibit approximately 90% of the protease activity of DeltaNS3 and MBP-NS3 (full-length of NS3 fused with maltose binding protein). In addition, these aptamers inhibited approximately 70% of the MBP-NS3 protease activity in the presence of the NS4A peptide P41. G9-I aptamer appeared to be a noncompetitive inhibitor for DeltaNS3 with a Ki approximately 100 nM in the presence of P41. These results suggest that the pool of selected aptamers have potential as anti-HCV compounds. Mutational analysis of the G9-I aptamer demonstrated that the sequences required for protease inhibition are in stem I, stem III and loop III of the aptamer. These regions include the conserved sequence GA(A/U)UGGGAC. 相似文献
16.
Combining SELEX and the yeast three-hybrid system for in vivo selection and classification of RNA aptamers
下载免费PDF全文
![点击此处可从《RNA (New York, N.Y.)》网站下载免费的PDF全文](/ch/ext_images/free.gif)
König J Julius C Baumann S Homann M Göringer HU Feldbrügge M 《RNA (New York, N.Y.)》2007,13(4):614-622
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. 相似文献
17.
Tetracyclines (TCs) are a group of antibiotics comprising of a common tetracycline (TET) nucleus with variable X(1) and X(2) positions on 5 and 6 carbon atoms, such as oxytetracycline (OTC) and doxycycline (DOX). In this study, the tetracycline group specific (TGS) ssDNA aptamers were identified by modified SELEX method by employing tosylactivated magnetic beads (TMB) coated with OTC, TET, and DOX, respectively, as targets and counter targets. Twenty TGS-aptamers were selected, of which seven aptamers, designated as T7, T15, T19, T20, T22, T23, and T24, showed high affinity to the basic TET backbone (K(d)=63-483 nM). The specificity of these TGS-aptamers to structural analogues followed the order in which the TCs was employed during SELEX process (OTC>TET>DOX) except aptamer T22, which was highly specific to TET than OTC or DOX. Aptamers that were specific to one target molecule but fail to bind the other structurally related TCs were eliminated during counter selection steps. Three aptamers, T7, T19, and T23 contained palindromic consensus sequence motif GGTGTGG. The remaining TGS-aptamers showed many consensus sequences that are truncated forms of this palindrome forming mirror image or inverted sequences. For example, GTGG or its inverted form, GGTG motif was found in all TGS-aptamers. A consensus sequence motif TGTGCT or its truncated terminal T-residue was found in most TGS-aptamers, which is predicted to be essential for high affinity and group specificity. These TGS-aptamers have potential applications such as target drug delivery, and detection of TCs in pharmaceutical preparations and contaminated food products. 相似文献
18.
Djordjevic M 《Biomolecular engineering》2007,24(2):179-189
Systematic Evolution of Ligands by EXponential enrichment (SELEX) is an experimental procedure that allows extraction, from an initially random pool of oligonucleotides, of the oligomers with a desired binding affinity for a given molecular target. The procedure can be used to infer the strongest binders for a given DNA or RNA binding protein, and the highest affinity binding sequences isolated through SELEX can have numerous research, diagnostic and therapeutic applications. Recently, important new modifications of the SELEX protocol have been proposed. In particular, a modification of the standard SELEX procedure allows generating a dataset from which protein-DNA interaction parameters can be determined with unprecedented accuracy. Another variant of SELEX allows investigating interactions of a protein with nucleic-acid fragments derived from the entire genome of an organism. We review here different SELEX-based methods, with particular emphasis on the experimental design and on the applications aimed at inferring protein-DNA interactions. In addition to the experimental issues, we also review relevant methods of data analysis, as well as theoretical modeling of SELEX. 相似文献
19.
We have applied SELEX (Systematic Evolution of Ligands by EXponential enrichment), a combinatorial method that employs biopolymers for drug discovery, to identify single stranded DNA sequences able to bind L-Tyrosinamide, a simple mimic of Tyrosine, an amino acid essential to the catalytic activity of several enzymes of pharmaceutical interest. After 15 SELEX cycles using L-Tyrosinamide immobilized on an affinity chromatography column, the percentage of aptamers specifically eluted from the affinity column with free L-Tyrosinamide was 55% of the total. Aptamers were subcloned and sequenced, allowing the identification of a highly conserved consensus sequence, and showed a K(d) value for L-Tyrosinamide of 45 microM. The identified aptamer sequence will constitute the basis for further in vitro evolution protocols and structure-based drug design. 相似文献
20.
Huiyu Liang Yusheng Shi Zhewen Kou Yonghua Peng Wenjun Chen Xiaowen Li Shuji Li Ying Wang Fang Wang Xingmei Zhang 《PloS one》2015,10(10)
An initial step in amyloid-β (Aβ) production includes amyloid precursor protein (APP) cleavage via β-Site amyloid precursor protein cleaving enzyme 1 (BACE1). Increased levels of brain Aβ have been implicated in the pathogenesis of Alzheimer’s disease (AD). Thus, β-secretase represents a primary target for inhibitor drug development in AD. In this study, aptamers were obtained from combinatorial oligonucleotide libraries using a technology referred to as systematic evolution of ligands by exponential enrichment (SELEX). A purified human BACE1 extracellular domain was used as a target to conduct an in vitro selection process using SELEX. Two DNA aptamers were capable of binding to BACE1 with high affinity and good specificity, with Kd values in the nanomolar range. We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model. We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment. Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media. Furthermore, Western blot analysis indicated that sAPPβ expression significantly decreased in the A1 treated versus control groups. These findings support the preliminary feasibility of an aptamer evolved from a SELEX strategy to function as a potential BACE1 inhibitor. To our knowledge, this is the first study to acquire a DNA aptamer that exhibited binding specificity to BACE1 and inhibited its activity. 相似文献