核酸适配体在病原生物学中的应用研究进展

核酸适配体是利用指数富集配体系统进化（Ｓｙｓｔｅｍａｔｉｃ ｅｖｏｌｕｔｉｏｎ ｏｆ ｌｉｇａｎｄｓ ｂｙ ｅｘｐｏｎｅｎｔｉａｌ ｅｎｒｉｃｈ ｍｅｎｔ，ＳＥＬＥＸ）技术筛选得到的能特异性识别和结合靶标的寡核苷酸片段，具有高亲和力和高特异性等特 点。 近年来，核酸适配体作为一种新型检测和治疗工具，已广泛应用于病原体检测、疾病诊断和治疗、新型 药物研发等生物医学领域。 本文对核酸适配体在病原生物学中的应用研究进展进行简要综述。     

寄生蠕虫外泌体及其功能的研究进展

外泌体是一类由多种活细胞分泌的双层囊泡状小体,含有多种重要生物活性物质,如蛋白质、脂质和核酸等,参与介导细胞间的信息交流、免疫调节等重要生理过程。寄生虫来源的外泌体在寄生虫-宿主的相互作用中发挥重要作用,可通过各种机制调节宿主的免疫应答,实现对宿主细胞功能的调控,是寄生虫与寄生虫、寄生虫与宿主相互作用的重要媒介。本文就近年来外泌体在吸虫、绦虫和线虫等寄生蠕虫中感染致病、免疫调控和免疫逃避等方面的研究作一综述,为研究寄生蠕虫外泌体及其功能提供参考。     

外泌体miRNA在寄生虫中的进展

外泌体(exosomes)是由多种细胞分泌的膜囊泡,是细胞信息交流中的重要介质,因其含有携带遗传信息的核酸从而起着重要的调节作用。研究表明寄生虫可利用外泌体作为细胞间通讯及与宿主联系的工具,调节宿主的免疫系统,相互转移传递致病因子、药物抗性基因和分化因子,对疾病的发生发展具有重要作用。目前许多报道表明外泌体中的miRNA在调控虫体发育、介导相互作用及致病中都发挥了重要作用。本文就寄生虫外泌体miRNA的来源及构成、对寄生虫生长发育作用等生物学功能研究,以加深对寄生虫基因表达调控机制的理解,从而对寄生虫致病的分子机制研究提供理论基础。     

核酸疫苗——寄生虫疫苗的新希望

新近迅速发展起来的核酸疫苗为正面临困境的寄生虫疫苗尤其是疟疾、血吸虫病疫苗的研制带来了新的希望。核酸疫苗既具有重组亚单位疫苗的安全性,又具有减毒活疫苗诱导全方位免疫应答的高效的持久性。本文对核酸疫苗的研究动态、作用机制及应用前景,特别是抗寄生虫感染等方面进行了阐述和探讨。     

SELEX法筛选鼻咽癌核酸适配子的研究

目的建立鼻咽癌细胞核酸适配子库,为鼻咽癌的分子诊断、生物治疗奠定基础。方法体外合成长度为78个核苷酸的随机DNA文库,采用SELEX技术,以正常鼻咽上皮细胞为靶标进行消减筛选,以EB病毒阳性低分化鼻咽癌细胞为靶标进行10轮筛选。采用流式细胞仪检测判断核酸适配子库的特异性和亲合力,Clustal X软件分析适配子序列。结果流式细胞仪检测显示亚文库与靶细胞的结合能力随筛选轮数的增加荧光强度增强。聚类分析显示适配子可以分为3个家族,1、2家族具有保守序列。结论 EB病毒阳性鼻咽癌细胞适配子库初步建立成功,可筛选到具有较高亲和力和特异性的核酸适配子;本研究为鼻咽癌的分子诊断、生物治疗奠定了基础。     

外泌体及其他细胞外囊泡在寄生虫与寄生虫病 研究中的进展

外泌体（Exosome）是一种由大多数内源细胞分泌的微小膜泡，其中细胞外囊泡（Extracellular vesicles，EVs）是细胞特异性分泌的含有蛋白质、核酸和脂类等具有功能活性物质的膜泡，参与细胞间物质与信息传递，在机体免疫、新陈代谢以及肿瘤生长、转移和耐药等方面发挥作用。本文就近年来外泌体在寄生虫与寄生虫病研究中的进展作一综述。 ［关键词］     

外泌体及其他细胞外囊泡在寄生虫与寄生虫病 研究中的进展

外泌体（Exosome）是一种由大多数内源细胞分泌的微小膜泡，其中细胞外囊泡（Extracellular vesicles，EVs）是细胞特异性分泌的含有蛋白质、核酸和脂类等具有功能活性物质的膜泡，参与细胞间物质与信息传递，在机体免疫、新陈代谢以及肿瘤生长、转移和耐药等方面发挥作用。本文就近年来外泌体在寄生虫与寄生虫病研究中的进展作一综述。 ［关键词］     

核酸疫苗—寄生虫疫苗的新希望

新近迅速发展起来的核酸疫苗为正面临困境的寄生虫疫苗尤其是疟疾，血吸虫疫苗的研制带来了新的希望。核酸疫苗既具有重组亚单位疫苗的安全性，又具有减毒活疫苗诱导全方位免疫应签的高效的持久性。本文对核酸疫苗的研究动态，作用机制及前景，特别是抗寄生虫感染等方面进行了阐述和探讨。     

核酸适配子在消化系统肿瘤诊治的应用研究进展

核酸适配子是一类寡核苷酸配基,由指数富集配体系统进化技术从寡核苷酸文库中筛选合成所得.核酸适配子的配体种类丰富,其亲和力及特异性较蛋白抗体高、性质稳定及合成简便,有望成为生物医药领域的花魁.核酸适配子在疾病尤其是肿瘤诊治的应用中已表现出很大潜力.此文就核酸适配子在消化系肿瘤诊治的应用研究进展作一综述.     

核酸定量扩增技术在寄生虫及病原微生物诊断中的研究进展

在感染性疾病中针对寄生虫及微生物病原体的核酸检测与病原学检测具有同等的价值,而核酸定量扩增技术不仅可定性诊断,还可用于各种感染性疾病的感染度测定、疗效考核和愈后评价等.该技术已成为近年来分子诊断研究的热点之一.该文对核酸定量扩增技术在感染性疾病诊断中的研究进展作一综述.     

The potential of aptamers as anticoagulants

Useful additional options for anticoagulant therapy have been introduced over the last 15 years, including low-molecular-weight heparins and direct thrombin inhibitors. Despite these impressive advances, a need for safer effective anticoagulants remains. Aptamers represent a therapeutic modality that has the potential to address this unmet need. Aptamers are small nucleic acid molecules that function as direct protein inhibitors, much like monoclonal antibodies. Aptamers are delivered by parenteral administration, can be formulated to possess a very short or sustained half-life, and are purported to be nonimmunogenic. Perhaps most relevant to the development of safer anticoagulant therapies, recent studies have shown that antidotes can be rationally designed to control the pharmacologic effects of aptamers in vivo, paving the way for a new class of antidote-controlled therapeutics. This review discusses the limitations of current anticoagulant therapies, the properties of aptamers and how these properties can be exploited to address the unmet needs within this therapeutic class, and the progress to date in developing new aptamer-based anticoagulant therapies.     

Targeted modification and transportation of cellular proteins

Peptide aptamers are proteins selected from combinatorial libraries that display conformationally constrained variable regions. Peptide aptamers can disrupt specific protein interactions and thus represent a useful method for manipulating protein function in vivo. Here, we describe aptamer derivatives that extend the range of functional manipulations. We isolated an aptamer with increased affinity for its Cdk2 target by mutagenizing an existing aptamer and identifying tighter binding mutants with calibrated two-hybrid reporter genes. We used this and other anti-Cdk2 aptamers as recognition domains in chimeric proteins that contained other functional moieties. Aptamers fused to the catalytic domain of a ubiquitin ligase specifically decorated LexA-Cdk2 with ubiquitin moieties in vivo. Aptamers against Cdk2 and another protein, Ste5, that carried a nuclear localization sequence transported their targets into the nucleus. These experiments indicate that fusion proteins containing aptameric recognition moieties will be useful for specific modification of protein function in vivo.     

Screening of aptamers and their potential application in targeted diagnosis and therapy of liver cancer

Aptamers are a class of single oligonucleotide molecules(DNA or RNA)that are screened from random DNA or RNA oligonucleotide chain libraries by the systemic evolution of ligands by exponential enrichment technology.The selected aptamers are capable of specifically binding to different targeting molecules,which is achieved by the three-dimensional structure of aptamers.Aptamers are similar in function to monoclonal antibodies,and therefore,they are also referred to as"chemical antibodies".Due to their high affinity and specificity and low immunogenicity,aptamers are topics of intense interest in today's biological targeting research especially in tumor research.They not only have high potential for clinical advances in tumor targeting detection but also are highly promising as targeted tumor drug carriers for use in tumor therapy.Various experimental studies have shown that aptamer-based diagnostic and therapeutic methods for liver cancer have great potential for application.This paper summarizes the structure,characteristics,and screening methods of aptamers and reviews the recent research progress on nucleic acid aptamers in the targeted diagnosis and treatment of liver cancer.     

Translating Nucleic Acid Aptamers to Antithrombotic Drugs in Cardiovascular Medicine

Nucleic acid aptamers offer several distinct advantages for the selective inhibition of protein targets within the coagulation cascade. A highly attractive feature of aptamers as antithrombotics is their ability to encode for complementary “controlling agents” which selectively bind to and neutralize their active counterparts via Watson–Crick base pairing or, in a less selective and clinically characterized manner, cationic polymers that can counteract the activity of an aptamer or free/protein-complexed nucleic acid. The former property allows aptamer-based antithrombotic therapies to be administered with a goal of selective, high intensity target inhibition, knowing that rapid drug reversal is readily available. In addition, by purposefully varying the ratio of active agent to a specific controlling agent administered, the intensity of antithrombotic therapy can be regulated with precision according to patient needs and the accompanying clinical conditions. REG1, currently undergoing phase 2B clinical investigation, consists of an RNA aptamer (RB006; pegnivacogin) which targets factor IXa and its complementary controlling agent (RB007; anivamersen). Aptamers directed against other serine coagulation proteases, some with and some without parallel controlling agents, have been designed. Aptamers directed against platelet surface membrane receptor targets are in preclinical development. The following review offers a contemporary summary of nucleic acid aptamers as a translatable platform for regulatable antithrombotic drugs expanding the paradigm of patient- and disease-specific treatment in clinical practice.     

Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing

We describe the integration of microfluidic selection with high-throughput DNA sequencing technology for rapid and efficient discovery of nucleic acid aptamers. The Quantitative Selection of Aptamers through Sequencing method tracks the copy number and enrichment-fold of more than 10 million individual sequences through multiple selection rounds, enabling the identification of high-affinity aptamers without the need for the pool to fully converge to a small number of sequences. Importantly, this method allows the discrimination of sequences that arise from experimental biases rather than true high-affinity target binding. As a demonstration, we have identified aptamers that specifically bind to PDGF-BB protein with K_d < 3 nM within 3 rounds. Furthermore, we show that the aptamers identified by Quantitative Selection of Aptamers through Sequencing have ∼3–8-fold higher affinity and ∼2–4-fold higher specificity relative to those discovered through conventional cloning methods. Given that many biocombinatorial libraries are encoded with nucleic acids, we extrapolate that our method may be extended to other types of libraries for a range of molecular functions.     

泡沫细胞靶向适配子的体外筛选

目的筛选巨噬细胞源性泡沫细胞的寡核苷酸适配子,为动脉粥样硬化的靶向治疗提供实验依据。方法以80 mg/L氧化型低密度脂蛋白孵育THP-1巨噬细胞72 h,建立泡沫细胞模型;利用指数富集配基的系统进化技术,从体外合成的随机单链DNA文库中筛选特异性寡核苷酸适配子;荧光显微镜观察寡核苷酸文库与泡沫细胞结合的特异性;克隆、测序确定适配子的序列并进行一级结构和二级结构分析。结果通过油红O染色和高效液相色谱分析,确定成功建立泡沫细胞模型;经过18轮的循环筛选,寡核苷酸文库仅与泡沫细胞结合,不再结合巨噬细胞、血管平滑肌细胞。测序鉴定出的所有适配子序列可以分为12个家族,没有共同的同源序列。二级结构分析表明,适配子主要形成茎环结构,这可能是适配子与巨噬细胞源性泡沫细胞结合的结构基础。结论利用复合靶指数富集配基的系统进化技术成功筛选出巨噬细胞源性泡沫细胞的寡核苷酸适配子。     

Xeno-Nucleic Acid (XNA) 2’-Fluoro-Arabino Nucleic Acid (FANA) Aptamers to the Receptor-Binding Domain of SARS-CoV-2 S Protein Block ACE2 Binding

The causative agent of COVID-19, SARS-CoV-2, gains access to cells through interactions of the receptor-binding domain (RBD) on the viral S protein with angiotensin-converting enzyme 2 (ACE2) on the surface of human host cells. Systematic evolution of ligands by exponential enrichment (SELEX) was used to generate aptamers (nucleic acids selected for high binding affinity to a target) to the RBD made from 2ʹ-fluoro-arabinonucleic acid (FANA). The best selected ~79 nucleotide aptamers bound the RBD (Arg319-Phe541) and the larger S1 domain (Val16-Arg685) of the 1272 amino acid S protein with equilibrium dissociation constants (K_D,app) of ~10–20 nM, and binding half-life for the RBD, S1 domain, and full trimeric S protein of 53 ± 18, 76 ± 5, and 127 ± 7 min, respectively. Aptamers inhibited the binding of the RBD to ACE2 in an ELISA assay. Inhibition, on a per weight basis, was similar to neutralizing antibodies that were specific for RBD. Aptamers demonstrated high specificity, binding with about 10-fold lower affinity to the related S1 domain from the original SARS virus, which also binds to ACE2. Overall, FANA aptamers show affinities comparable to previous DNA aptamers to RBD and S1 protein and directly block receptor interactions while using an alternative Xeno-nucleic acid (XNA) platform.     

Isolation of high-affinity GTP aptamers from partially structured RNA libraries

Aptamers, RNA sequences that bind to target ligands, are typically isolated by in vitro selection from RNA libraries containing completely random sequences. To see whether higher-affinity aptamers can be isolated from partially structured RNA libraries, we selected for aptamers that bind GTP, starting from a mixture of fully random and partially structured libraries. Because stem-loops are common motifs in previously characterized aptamers, we designed the partially structured library to contain a centrally located stable stem-loop. We used an off-rate selection protocol designed to maximize the enrichment of high-affinity aptamers. The selection produced a surprisingly large number of distinct sequence motifs and secondary structures, including seven different aptamers with K(d)s ranging from 500 to 25 nanomolar. The engineered stem-loop was present in the three highest affinity aptamers, and in 12 of 13 independent isolates with a single consensus sequence, suggesting that its inclusion increased the abundance of high-affinity aptamers in the starting pool.     

Aptamers in Diagnostics and Treatment of Viral Infections

Aptamers are in vitro selected DNA or RNA molecules that are capable of binding a wide range of nucleic and non-nucleic acid molecules with high affinity and specificity. They have been conducted through the process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). It serves to reach specificity and considerable affinity to target molecules, including those of viral origin, both proteins and nucleic acids. Properties of aptamers allow detecting virus infected cells or viruses themselves and make them competitive to monoclonal antibodies. Specific aptamers can be used to interfere in each stage of the viral replication cycle and also inhibit its penetration into cells. Many current studies have reported possible application of aptamers as a treatment or diagnostic tool in viral infections, e.g., HIV (Human Immunodeficiency Virus), HBV (Hepatitis B Virus), HCV (Hepatitis C Virus), SARS (Severe Acute Respiratory Syndrome), H5N1 avian influenza and recently spread Ebola. This review presents current developments of using aptamers in the diagnostics and treatment of viral diseases.