荧光核酸适配体：核酸酶学分析新机遇

DNA和RNA的合成与降解等核酸代谢过程是维持生命体生长发育、新陈代谢、遗传变异、个体衰老等生命过程的基础代谢单元，广泛参与机体生命活动的全过程。核酸代谢相关酶活性对于维持细胞内环境的稳定性至关重要，其活性的改变可能会引起多种疾病的发生与发展。核酸代谢相关酶已成为多种疾病研究的重要靶点，也是生物技术和生物工程领域中不可或缺的重要工具，如聚合酶链反应、定点诱变、分子克隆和DNA测序等。因此，核酸代谢是所有核酸研究和相关生命科学领域研究的基础。本文将介绍核酸代谢的酶学分析常用方法，并聚焦于操作简单、实时快速的荧光法，按照荧光法的检测原理、发展历史和应用进行分类阐述与比较，并对核酸酶学分析工具未来研究与发展方向进行展望。     

细胞特异性核酸适配体筛选新策略的研究进展

核酸适配体(aptamer)是在体外采用指数富集的配基系统进化技术(systematic evolution of ligands by exponential enrichment, SELEX)从人工合成的随机寡核苷酸文库中筛选得到的一段寡核苷酸序列(RNA或DNA),能折叠成特定的三维空间结构同靶物质进行高特异性与高亲和性的结合。近年来,以全细胞为靶标筛选(cell-SELEX)获得的核酸适配体在疾病相关的领域有很大的应用潜力,尤其在细胞分子的识别、生物标志物的发现等方面,但cell-SELEX的过程复杂、难度大及获得的核酸适配体性能不佳等问题仍然制约着细胞特异性核酸适配体的进一步发展,如何高效地筛选获得核酸适配体是其应用的关键。本文总结了目前在cell-SELEX技术基础上发展起来的新方法、新策略及核酸适配体在肿瘤研究中的应用,望为相关科研人员的研究提供参考。     

应用双向热循环消减SELEX技术筛选肺癌血清核酸适配体

肺癌是发病率和死亡率较高的恶性肿瘤。现阶段,用于肺癌早期诊断的血清肿瘤标志物因其特异性与敏感性均较低,严重影响肺癌的临床诊断和治疗。本文用双向热循环消减指数富集的配基进化(systematic evolution of ligands by exponential enrichment, SELEX)技术,筛选肺癌和非癌血清标志物的核酸适配体,建立肺癌的检测方法,提高诊断和治疗效率。实验用环氧基琼脂磁珠为筛选介质,以非癌混合血清、肺癌混合血清作为双向靶标。应用热循环消减SELEX技术,经19轮筛选分别获得非癌和肺癌血清的特异性核酸适配体。通过高通量测序,得到 40条非癌核酸适配体序列和 41条肺癌核酸适配体序列。从肺癌与非癌血清特异性核酸适配体序列中分别挑选出高丰度的 4条序列,合成后制成检测试剂,经临床血清验证,阳性率为 90%。该检测方法检测灵敏度高,为肺癌的早期诊断和治疗提供了新的分子识别元件。     

双向热循环消减-SELEX方法筛选肝癌血清核酸适配体

肝癌位于我国肿瘤死亡率第2位,生存率较低。目前用于肝癌早期诊断的临床检查及血清肿瘤标志物检测的特异性与敏感性均较低,不能满足肝癌早期诊断和治疗的需要。核酸适配体与靶标分子结合的灵敏度高、特异性强,有巨大的临床诊断和治疗应用前景。本文利用双向热循环消减指数富集的配基系统进化（systematic evolution of ligands by exponential enrichment, SELEX）技术,分别以肝癌血清和健康人血清为靶标,经过19轮筛选,获得了肝癌血清特异性核酸适配体序列1 000余条,以及健康人血清特异性核酸适配体序列1 000余条,并从中各挑取了1条高丰度适配体序列,分别命名为Tc1和Tn1。采取了50例肝癌病人血清和50例健康人血清,对适配体Tc1和Tn1与靶标血清的结合特异性进行了检测。结果显示,Tc1和Tn1对两种靶标血清的检出率分别为92%和94%。说明Tc1可特异性与肝癌血清结合,Tn1可特异性与健康人血清结合。肝癌血清特异性核酸适配体的筛选获得,将为建立基于核酸适配体的肝癌血清检测新方法奠定基础。     

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。     

应用双向热循环消减SELEX技术筛选胃癌血清核酸适配体

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。     

Selection,Characterization and Application of Nucleic Acid Aptamers for the Capture and Detection of Human Norovirus Strains

Human noroviruses (HuNoV) are the leading cause of acute viral gastroenteritis and an important cause of foodborne disease. Despite their public health significance, routine detection of HuNoV in community settings, or food and environmental samples, is limited, and there is a need to develop alternative HuNoV diagnostic reagents to complement existing ones. The purpose of this study was to select and characterize single-stranded (ss)DNA aptamers with binding affinity to HuNoV. The utility of these aptamers was demonstrated in their use for capture and detection of HuNoV in outbreak-derived fecal samples and a representative food matrix. SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used to isolate ssDNA aptamer sequences with broad reactivity to the prototype GII.2 HuNoV strain, Snow Mountain Virus (SMV). Four aptamer candidates (designated 19, 21, 25 and 26) were identified and screened for binding affinity to 14 different virus-like particles (VLPs) corresponding to various GI and GII HuNoV strains using an Enzyme-Linked Aptamer Sorbant Assay (ELASA). Collectively, aptamers 21 and 25 showed affinity to 13 of the 14 VLPs tested, with strongest binding to GII.2 (SMV) and GII.4 VLPs. Aptamer 25 was chosen for further study. Its binding affinity to SMV-VLPs was equivalent to that of a commercial antibody within a range of 1 to 5 µg/ml. Aptamer 25 also showed binding to representative HuNoV strains present in stool specimens obtained from naturally infected individuals. Lastly, an aptamer magnetic capture (AMC) method using aptamer 25 coupled with RT-qPCR was developed for recovery and detection of HuNoV in artificially contaminated lettuce. The capture efficiency of the AMC was 2.5–36% with an assay detection limit of 10 RNA copies per lettuce sample. These ssDNA aptamer candidates show promise as broadly reactive reagents for use in HuNoV capture and detection assays in various sample types.     

多肽核酸

通过计算机模型设计出一种以肽骨架取代ＤＮＡ中脱氧核糖磷酸酯骨架的ＤＮＡ类似物．实验已证明其与互补寡核苷酸的结合具有高度的特异性．综述了其合成、杂交性及在分子生物学等方面的应用．     

反基因锁核酸体外阻断乙肝病毒前S2基因表达

目的:探讨针对乙肝病毒前S2基因同聚嘌呤区的锁核酸体外抑制细胞内病毒复制的作用.方法:针对乙肝病毒前S2基因同聚嘌呤区,分别设计合成锁核酸、硫代寡核苷酸、未修饰寡核苷酸及无关对照序列,以半乳糖配体介导转染HepG2.2.15细胞,采用荧光定量聚合酶链反应技术(FQ-PCR)、时间分辨免疫荧光技术(TRFIA)和酶联免疫法(ELISA)分别监测1、3、5和7d细胞培养上清液中HBV DNA、HBsAg和前S2抗原的含量;四甲基偶氮唑蓝(MTT)法检测锁核酸对细胞代谢的影响.结果:加入锁核酸后,对HBV DNA复制、HBsAg和前S2抗原表达均显示有较强的抑制作用,且抑制率随时间呈增高趋势,7d后抑制率分别达61.56％、68.18％和72.82％.各实验组与对照组比较差异均具有统计学意义(均P＜0.05).LNA对细胞代谢无明显影响.结论:针对乙肝病毒前S2基因同聚嘌呤区的反基因锁核酸,体外能有效抑制乙肝病毒的复制,既为乙肝病毒治疗提供有效靶位,也为反基因治疗提供理论和实验依据.     

研究报告: 基于分子对接和步进序列群的蓖麻毒素核酸适配体序列优化

目的 有效结合分子对接预测和表面等离子体共振实验评价技术，获得亲和力更强、序列最短的最优适配体。方法 针对前期筛选出的靶向蓖麻毒素的3条80 nt单链DNA适配体（L14、P3、L7），在明确各自二维随机区茎环序列与靶蛋白结合能力的基础上，以H-DOCK分子对接为指导，分别确定蓖麻毒素适配体随机区的最短结合单元，从而构建两端延长步进序列群，以表面等离子体共振技术测定序列群序列的亲和力和动力学参数，明确适配体的结合关键结构，从而筛选得到最优适配体。结果 3条全长适配体的随机区适配体L14r、P3r、L7r均可形成一定的茎环结构，其中L14r较L14的亲和力增强9倍、L7r增强2倍、P3r基本不变。对随机区适配体和蓖麻毒素进行分子对接，结果显示，L14r、P3r、L7r的对接分数值皆优于阴性序列40T，结合关键氨基酸个数分别为11、8、9个，存在距离小于5 ?的预测结合位点分别为20、12、15个，具有良好的与蓖麻毒素的结合能力。进一步明确了蓖麻毒素活性口袋所容纳的适配体最短结合单元L14rm、P3rm、L7rm的序列构成，在此基础上构建出两端延长步进序列群。针对该步进群，基于结合关键氨基酸个数、结合位点个数、对接得分等参数的变化和表面等离子体共振测定结果筛选出最优适配体。所获得的最优适配体L14rm、L7rm-2亲和力继续增强了1~2倍。结论 随机区适配体能有效地与蓖麻毒素结合，较之全长适配体亲和力更强，分子对接结合步进序列群设计，仅使用17条序列，便有效获得了3条最优适配体并明确其结合作用。3条结合蓖麻毒素的最优适配体——L14rm、P3r、L7rm-2的K_D值分别为（64±30）、（167±19）、（120±1）nmol/L，亲和力提高到全长适配体的14、1、4倍。     

Peptide-Templated Nucleic Acid Ligation

Abstract Short oligonucleotide and peptide replicators have been described. To determine whether cross-replication could have occurred between such systems, we have attempted to show that peptides can specifically template the ligation of nucleic acids. A complex between a 35-mer anti-Rev RNA aptamer and a 17-mer arginine-rich motif (ARM) peptide from the HIV-1 Rev protein served as a model system. Aptamer half-molecules were activated for ligation via two activation chemistries, representing two distinct kinetic possibilities for early replicators. Cyanogen bromide activation was transient relative to oligonucleotides that terminated with a 5′-iodine and a 3′phosphorothioate, respectively. The Rev ARM specifically enhanced the degree or rate of ligation by both methods: there was a 10-fold increase in the production of full-length aptamer in the presence of cyanogen bromide and a 5.9- to 7.6-fold enhancement in the rate of ligation for stably activated aptamer half-molecules. These results support the possibility that life could have originated with peptide replicators and transitioned to nucleic acid replicators or that peptide and nucleic acid replicators could have been interdependent.     

Fluorinated Peptide Nucleic Acid

Abstract

The fluorinated olefinic peptide nucleic acid analogue (F-OPA) monomer containing the base thymine was synthesised in 13 steps. PNAs containing this unit were prepared and their pairing properties assessed by means of UV-melting experiments.     

Nanoparticles and Nanomotors Modified by Nucleic Acids Aptamers for Targeted Drug Delivery

Russian Journal of Bioorganic Chemistry - Targeted drug delivery is a new method in the therapy of various diseases, especially cancer. Typically the therapeutic drug is encapsulated into the...     

核酸纯化柱提取核酸定量检测丙型肝炎病毒RNA的临床应用

目的:探究核酸纯化柱提取核酸定量检测血浆标本中丙型肝炎病毒核糖核酸(HCV-RNA)的临床应用效果。方法:将2013年8-11月期间我院600例抗-HCV阳性丙肝患者的样本,按随机字数表法分为研究组对照组各300例,分别采用核酸纯化柱和酚-氯仿提取法检测,采用实时荧光定量聚合酶链反应(FQ-PCR)技术定量检测两组HCV-RNA水平。结果:研究组检测阳性率为87.67%(263/300),显著高于和对照组的54.0%(162/300),差异有统计学意义(X2=82.296,P=0.000);两组HCV-RNA检测水平差异无统计学意义(u=1.721,P=0.067)。结论:核酸柱提法定量检测血浆HCV-RNA操作简单、快速,分离效率高,容易掌握,值得临床推广。     

Berenil Complexation with a Nucleic Acid Triple Helix

Abstract

The interaction of berenii molecule, a minor groove binding drug, with T-A-T triple helix and A-T double helix was studied using circular dichroism spectroscopy and thermal denaturation. The triple helix was made by an oligonucleotide (dA)₁₂?x-(dT)₁₂?x-(dT)₁₂, where x is a hexaethylene glycol chain bridged between the 3′ phosphate of one strand and the 5′ phosphate of the following strand. This oligonucleotide is able to fold back on itself to form a very stable triplex. Circular dichroism spectroscopy demonstrates that berenil can bind to the triple helical structure. Spectral analysis shows that in the same ionic strength the drug bound to a double-stranded structure exhibits a conformation and an environment close to those observed in triple-stranded structure. The influence of the ionic strength on the interaction between the berenil molecule and the 36-mer is clearly demonstrated. We showed that when no NaCl salt is added in the buffer the triplex form of (dA)₁₂?x-(dT)₁2-x-(dT)₁₂ is stabilized by berenil whereas it is destabilized slightly by the dye when NaCl concentration is 1 M.     

Liquid Phase Synthesis of Peptide Nucleic Acid (or Polyamide Nucleic Acid) Dimers

Abstract

The liquid phase synthesis of “polyamide nucleic acid” (PNA) dimers containing the purine nucleic acid bases adenine and guanine has been achieved in good yields. This strategy was elaborated in order to circumvent difficult direct coupling of protected PNA monomers. This method can be applied to the liquid phase synthesis of short protected polyPNAs fragments, which can then selectively be deprotected.     

Nucleobase Modified Peptide Nucleic Acid

Abstract

The Pd⁰/Cu^I catalyzed cross-coupling of terminal alkynes onto peptide nucleic acid monomers or submonomers bearing iodinated nucleobases has been utilized as a route to base-modified oligomers. Both 5-iodouracil and 5-iodocytosine derivatives undergo the cross-coupling to give the expected products in moderate to good yields. However, depending on the particular substrates and reaction conditions, the cross-coupling may be followed by a ring closing reaction to give the fluorescent furano- and pyrrolo-fused uracil and cytosine derivatives, respectively.