基于适体的石英晶体微天平传感器的研究进展

适体(Aptamer)是通过指数富集配体系统进化技术(Systematic evolution of ligands by exponential enrichment,SELEX)从人工合成的随机单链寡核苷酸文库里筛选出来的短链寡核苷酸序列,具有分子量小、结构简单、易进行修饰、靶标范围广泛,并且与靶标分子之间具有高特异性和高亲和力等特点。相应地,适体的这些独特的性质可用于制备各种不同的传感器,根据各种传感器的不同原理,本文着重概述了常用于检测适体与靶标之间亲和力的电化学生物传感器、光学生物传感器和压电晶体传感器。这3种方法的检测都具有检测时间短和检测限低的优势。其中压电晶体传感器又称石英晶体微天平(Quartz crystal microbalance,QCM),除了在SELEX技术中可应用于表征候选适体的靶向能力外,还可用于构建高灵敏度和高特异性的适体石英晶体传感器。简要介绍了基于适体的石英晶体微天平传感器基本原理,对近年来QCM在表征和检测适体与其靶标,包括小分子、离子、蛋白质、细胞、细菌和病毒等物质相互作用的研究现状进行综述,总结分析了QCM技术的优缺点。旨在为适体的筛选以及适体在基础研究、临床诊断和疾病治疗中的进一步应用提供参考。     

核糖开关的研究及应用

核糖开关是一类自然界中天然存在的适配子,通过结合小分子代谢物调控基因的表达。它位于特定的mRNA非编码区,可以不依赖任何蛋白质因子而直接结合代谢物并发生构象变化,在转录和翻译水平上参与调控生物的基本代谢途径。目前已知核糖开关不仅广泛存在于细菌的代谢相关基因中,还存在于某些真菌和植物中。对核糖开关的深入研究将为基因功能研究、生物传感器研发以及新型抗菌药物开发等提供新的途径。     

核糖核酸调节子的构建与即时检测中的应用*

合成生物学专注于可重复利用模块和元件的工程化设计,并在生物系统中表现出良好的行为和功能。在无细胞蛋白表达系统中,核糖核酸调节子作为即时检测中的重要传感元件,通过靶标分子的诱导使其自身结构发生变化,进而调控下游基因的表达。系统介绍不同类型的核糖核酸调节子及其作用原理,包括一代核糖核酸调节子、toehold开关、功能拓展的核糖核酸调节子和核糖开关。详细阐明构建核糖核酸调节子的设计-测试-分析过程:计算机辅助设计、基因表达测试和结构功能化分析。汇总基于核糖核酸调节子的体外即时检测应用,重点总结toehold开关介导的病原菌核酸检测和核糖开关参与的小分子检测。讨论当前无细胞即时检测的特点、挑战和发展趋势,为开发新型核糖核酸调节子和即时检测工具提供思路和参考。     

化学蛋白质组学技术在药物靶标鉴定中的应用

药物开发过程面临多重挑战,而靶标确证是其中的重要一环。如何运用多种研究方法发现和确认小分子药物的靶标是目前研究人员的主要工作内容之一。化学蛋白质组学整合了细胞生物学、合成化学和生物质谱等多门学科,为药物的靶标筛选提供了新平台。本文对近年来发展的基于生物质谱的化学蛋白质组学药物靶标鉴定技术进行了总结,结合具体应用分析其优缺点,并对该类技术的发展和应用进行总结和展望。     

基于核糖开关的新型基因表达调控系统的应用

核糖开关是能对细胞环境的改变做出反应的顺式作用元件,通过改变自身的构象实现对基因表达的调控。基于核糖开关调控方式简洁,无需蛋白质参与,响应迅速,且自身片段小,结构简单,易于设计和改造等特性使其在生物医学领域体现出诸多应用优势。对核糖开关的结构,调节机理以及这种新型基因表达调控系统在基因治疗、抗生素新靶点的开发、病毒疫苗的安全控制、新型核糖选择器和生物体内传感器的应用进行了综述,旨为启示我国核糖开关的新型应用。     

核糖开关及其在抗菌药物方面的研究进展

核糖开关是一类与核酸、氨基酸、金属离子、糖类衍生物以及辅酶等特异性配体结合的RNA元件,它与配体结合后通过调控相应下游的基因表达起到控制细胞生命及活动的作用。目前核糖开关是基因调控方面的研究热点,应用于大量筛选工程菌株、构建新型生物传感器以及作为抗菌药作用的新靶点。综述了几种主要的核糖开关(如:嘌呤核糖开关、赖氨酸核糖开关、环二鸟苷酸核糖开关、glm S核糖开关、TPP核糖开关、FMN核糖开关等)在抗菌药物靶点方面的研究进展。     

基于表面等离子共振的适配体传感器应用研究进展

基于表面等离子共振的适配体传感器是利用适配体进行高特异性、高灵敏度、高通量检测的新型生物传感器。我们在简要阐述适配体的筛选方法、偶联技术及适配体传感器工作原理的基础上,结合最新的研究结果,对基于表面等离子共振的适配体传感器在生物活性小分子检测、传染病检测、肿瘤标志物检测、食品安全监测等方面的应用研究进展进行了综述。     

核糖开关的结构和配体结合规律

核糖开关是一种RNA固有的基因表达调控系统。近年来,核糖开关的应用受到科研工作者的广泛关注,其结构与功能的研究也越来越受到重视。核糖开关具有哪些结构特征和调控机制,它是如何识别目的配体,又怎样与目的配体紧密结合,全面了解这些机制将为探索新型核糖开关,设计人工高效核糖开关提供重要思路。本文对核糖开关结构特征和调节机制、适体筛选、配体结合规律进行简要介绍。     

核糖开关的结构和调控机理

一种新发现的RNA分子——核糖开关,通过感知代谢物浓度的变化调控目标基因的表达。它可以调整自身的结构直接结合代谢物小分子,而不需要蛋白因子的参与。在原核生物中发现了大量的核糖开关,在真核生物如植物和真菌中也发现了核糖开关。核糖开关由适体域和表达平台两个功能域组成,能在不同水平调控基因的表达,如转录终止、翻译起始、mRNA剪辑和加工。核糖开关不需要蛋白因子的参与,因此人们认为它可能是古代RNA世界的遗留物。核糖开关作为RNA传感器可以设计成一种基因控制元件,在未来的基因治疗方面可能具有很大的应用前景。     

药物靶标蛋白筛选的化学蛋白质组学研究进展

药物或生物活性物质通过与靶蛋白结合而发挥功能,研究表明,大多数药物具有多个作用靶点,药物靶标的发现有助于药物前体的筛选和作用机制的研究,同时对其耐药性等副作用的解决方案提供理论指导.基于生物质谱技术的蛋白质组学可对蛋白质进行高通量的定性定量分析,为药物靶标的筛选提供了全新的平台.本文综述了基于固载药物和游离药物模式的药物靶标蛋白筛选相关方法和应用研究的最新进展,为基于生物质谱技术的化学蛋白质组学研究提供参考.     

Interactions of the c-di-GMP riboswitch with its second messenger ligand

The c-di-GMP [bis-(3'-5')-cyclic dimeric guanosine monophosphate] riboswitch is a macromolecular target in the c-di-GMP second messenger signalling pathway. It regulates many genes related to c-di-GMP metabolism as well as genes involved in bacterial motility, virulence and biofilm formation. The riboswitch makes asymmetric contacts to the bases and phosphate backbone of this symmetric dinucleotide. The phylogenetics suggested and mutagenesis has confirmed that this is a flexible motif where variants can make alternative interactions with each of the guanine bases of c-di-GMP. A mutant riboswitch has been designed that can bind a related molecule, c-di-AMP, confirming the most important contacts made to the ligand. The binding kinetics reveal that this is a kinetically controlled riboswitch and mutations to the riboswitch lead to increases in the off-rate. This riboswitch is therefore flexible in sequence as well as kinetic properties.     

An energetically beneficial leader-linker interaction abolishes ligand-binding cooperativity in glycine riboswitches

Comprised of two aptamers connected by a short nucleotide linker, the glycine riboswitch was the first example of naturally occurring RNA elements reported to bind small organic molecules cooperatively. Earlier works have shown binding of glycine to the second aptamer allows tertiary interactions to be made between the two aptamers, which facilitates binding of a separate glycine molecule to the first aptamer, leading to glycine-binding cooperativity. Prompted by a distinctive protection pattern in the linker region of a minimal glycine riboswitch construct, we have identified a highly conserved (>90%) leader-linker duplex involving leader nucleotides upstream of the previously reported consensus glycine riboswitch sequences. In >50% of the glycine riboswitches, the leader-linker interaction forms a kink-turn motif. Characterization of three glycine ribsowitches showed that the leader-linker interaction improved the glycine-binding affinities by 4.5- to 86-fold. In-line probing and native gel assays with two aptamers in trans suggested synergistic action between glycine-binding and interaptamer interaction during global folding of the glycine riboswitch. Mutational analysis showed that there appeared to be no ligand-binding cooperativity in the glycine riboswitch when the leader-linker interaction is present, and the previously measured cooperativity is simply an artifact of a truncated construct missing the leader sequence.     

Systematic optimization of L‐tryptophan riboswitches for efficient monitoring of the metabolite in Escherichia coli

Riboswitches form a class of genetically encoded sensor‐regulators and are considered as promising tools for monitoring various metabolites. Functional parameters of a riboswitch, like dynamic or operational range, should be optimized before the riboswitch is implemented in a specific application for monitoring the target molecule efficiently. However, optimization of a riboswitch was not straightforward and required detailed studies owing to its complex sequence‐function relationship. Here, we present three approaches for tuning and optimization of functional parameters of a riboswitch using an artificial L‐tryptophan riboswitch as an example. First, the constitutive expression level was adjusted to control the dynamic range of an L‐tryptophan riboswitch. The dynamic range increased as the constitutive expression level increased. Then, the function of a riboswitch‐encoded protein was utilized to connect the regulatory response of the riboswitch to another outcome for amplifying the dynamic range. Riboswitch‐mediated control of the host cell growth enabled the amplification of the riboswitch response. Finally, L‐tryptophan aptamers with different dissociation constants were employed to alter the operational range of the riboswitch. The dose‐response curve was shifted towards higher L‐tryptophan concentrations when an aptamer with higher dissociation constant was employed. All strategies were effective in modifying the distinct functional parameters of the L‐tryptophan riboswitch, and they could be easily applied to optimization of other riboswitches owing to their simplicity.     

Computational analysis of riboswitch-based regulation

Advances in computational analysis of riboswitches in the last decade have contributed greatly to our understanding of riboswitch regulatory roles and mechanisms. Riboswitches were originally discovered as part of the sequence analysis of the 5′-untranslated region of mRNAs in the hope of finding novel gene regulatory sites, and the existence of structural RNAs appeared to be a spurious phenomenon. As more riboswitches were discovered, they illustrated the diversity and adaptability of these RNA regulatory sequences. The fact that a chemically monotonous molecule like RNA can discern a wide range of substrates and exert a variety of regulatory mechanisms was subsequently demonstrated in diverse genomes and has hastened the development of sophisticated algorithms for their analysis and prediction. In this review, we focus on some of the computational tools for riboswitch detection and secondary structure prediction. The study of this simple yet efficient form of gene regulation promises to provide a more complete picture of a world that RNA once dominated and allows rational design of artificial riboswitches. This article is part of a Special Issue entitled: Riboswitches.     

Rational design of SAM analogues targeting SAM-II riboswitch aptamer

Riboswitches are noncoding RNA elements embedded in 5′-untranslated region of many bacterial mRNAs regulating gene expression in response to essential metabolites. They are unique from other RNA targets because they have evolved to form specific structural receptors for the purpose of binding small molecular metabolites suggesting that structure-based rational drug design approach may be used in designing metabolite mimics targeting riboswitches. We have developed a fluorescence binding assay for SAM-II riboswitch aptamer and identified an S-adenosylmethionine (SAM) analogue that selectively binds to SAM-II riboswitch aptamer with comparable binding affinity to its native metabolite using structure-based design approach.     

The Ligand-Free State of the TPP Riboswitch: A Partially Folded RNA Structure

Riboswitches are elements of mRNA that regulate gene expression by undergoing structural changes upon binding of small ligands. Although the structures of several riboswitches have been solved with their ligands bound, the ligand-free states of only a few riboswitches have been characterized. The ligand-free state is as important for the functionality of the riboswitch as the ligand-bound form, but the ligand-free state is often a partially folded structure of the RNA, with conformational heterogeneity that makes it particularly challenging to study. Here, we present models of the ligand-free state of a thiamine pyrophosphate riboswitch that are derived from a combination of complementary experimental and computational modeling approaches. We obtain a global picture of the molecule using small-angle X-ray scattering data and use an RNA structure modeling software, MC-Sym, to fit local structural details to these data on an atomic scale. We have used two different approaches to obtaining these models. Our first approach develops a model of the RNA from the structures of its constituent junction fragments in isolation. The second approach treats the RNA as a single entity, without bias from the structure of its individual constituents. We find that both approaches give similar models for the ligand-free form, but the ligand-bound models differ for the two approaches, and only the models from the second approach agree with the ligand-bound structure known previously from X-ray crystallography. Our models provide a picture of the conformational changes that may occur in the riboswitch upon binding of its ligand. Our results also demonstrate the power of combining experimental small-angle X-ray scattering data with theoretical structure prediction tools in the determination of RNA structures beyond riboswitches.