分子灯塔设计核酸测定原理及应用

分子灯塔是一种在分子杂交基础上发展的一种荧光探针。这种探针５′端标记荧光物质,３′端连接荧光淬灭剂,而且在５′端和３′端设计互补碱基序列,使探针形成茎环结构。在检测核酸时具有快速、重复性好、灵敏度和特异性高、结果明确等优点。本文就分子灯塔的设计原则、分子特点和在核酸测定中的应用前景简要综述。     

单分子荧光共振能量转移技术

单分子荧光共振能量转移技术（single molecule fluorescence resonance energy transfer,smFRET）通过检测单个分子内的荧光供体及受体间荧光能量转移的效率,来研究分子构象的变化。在单分子探测技术发展之前,大多数的分子实验是探测分子的综合平均效应（ensemble averages）,这一平均效应掩盖了许多特殊的信息。单分子探测可以对体系中的单个分子进行研究,得到某一分子特性的分布状况,也可研究生物分子的动力学反应。介绍了近来单分子荧光共振能量转移技术的进展。     

甘露聚糖酶Man23的化学修饰及活性必需基团测定

用化学修饰剂NEM、二甲基溴化锍、EDC、DEPC、TNM、对硝基苯乙二醛、PMSF、TNBS对芽孢杆菌B23产生的甘露聚糖酶M an23进行化学修饰,并测定修饰反应的动力学参数关系。结果显示半胱氨酸、色氨酸(1个)和谷氨酸(或天冬氨酸)残基(2个)是酶活性的必需基团;组氨酸、酪氨酸、精氨酸、丝氨酸和赖氨酸残基均为非必需基团。双向电泳结果显示酶蛋白分子具有一个链内二硫键(Cys90-Cys110)。荧光光谱测定结果显示该酶最大吸收峰为336 nm。底物作用导致酶的发射光谱发生蓝移,说明色氨酸残基位于酶蛋白分子内部的疏水区。     

人类基因组DNA单核苷酸多态性的检测方法

单核苷酸多态性（SNP）作为新的遗传标记对基因定位及相关疾病的研究意义重大。本文对近年来9种SNP检测方法的原理、应用及优缺点,包括基于FRET原理的Taqman法和分子灯塔法;基于分子杂交技术的寡核苷酸连接分析、等位基因特异性寡核苷酸探针杂交法、动态等位基因特异性杂交法及DNA芯片法;及质谱法、变性－高压液相色谱法和单个碱基延伸标记法进行综述。     

蛋白质功能基团的改变与其生物活力之间的定量关系

蛋白质(包括酶)分子结构与功能的关系一直是分子生物学的核心问题之一.用各种方法改变蛋白质分子中侧链基团的性质观察对其生物活力的影响是研究这一问题的主要方法.蛋白质化学修饰的研究,迄今已有约60年的历史.自20年代末到60年代初的30余年内,这一研究一直停留在定性描述阶段,大量的实验结果不能进行定量的数据处理.因为在蛋白质分子中某类侧链基团在功能上虽有必需与非必需之分,但是在与某试剂起反应上往往是相同的,即它们都能与某一试剂起反应.所以,虽然从实验上可以测得随侧链基团的被修饰导致其生物活力下降的关系,但是在所测得的被修饰的基团中,既有必需的,也有非必需的.由于长期以来人们没有找到生物活力与必需基团之间的定量关系,也就无     

根据分子连接性指数和基团校正因子预测有机化合物在鱼体中的生物富集因子

研究了２３９种有机化合物的ＢＣＦ预测模型,结果显示根据分子连接性指数（ＭＣＩ）的线性模型不能准确计算及极性化合物的ＢＣＦ,也不适用于超疏水性化合物,在模型中引入基团校正因子后,极性经合物的计算误差明显降低。对数据进行曲线拟合,所得模型的估算精度进一步提高,最终确定以（＾０ｘ＾ｖ）＾２、（＾２ｘ＾ｖ）＾１／２、＾２ｘ、＾３ｘｃ、＾０ｘ＾ｖ及ＯＨ、ＮＨ２、ＮＯ２、ＮＣＯＯ等１０个基团校正团子为参数建立     

氨基酰化酶Ⅰ的必需半胱氨酸巯基

本文用DTNB和IAM修饰了猪肾氨基酰化酶Ⅰ的半胱氨酸巯基,用Koshland和邹承鲁作图法定量处理的结果都表明,酶分子中有二个巯基为酶的必需巯基。     

基于密度泛函叶绿素a分子活性的量子化学计算

在液相环境下,根据量子化学密度泛函(DFT)的B3LYP泛函在6-31G基组水平的计算结果,从叶绿素a分子的光谱特性、特征基团对前线轨道能量贡献率、激发态、Mulliken电荷分布等量化参数分析比较了其各特征基团的生物活性及吸收波长。结果表明:叶绿素a分子中的5个活性基团中,C(33)、O(34)所组成的酮羰基活性最强,卟啉环的活性次之,C(36)位置上的酯羰基和C(13)位置上的酯羰基及共轭碳碳双键的活性较弱;在发生HOMO→LUMO跃迁后,卟啉环转移的电荷变化总量为0.00012。而酮羰基得到的电荷变化,总量为-0.00062。     

光敏掩蔽基团在化学生物学中的应用

光敏掩蔽基团技术是通过运用一种由光子控制的光敏化合物,在光子激发后,被该化合物掩蔽处于惰性状态的生物活性分子重新被激活、释放以调节生理功能的化学生物学研究方法,它对生命活动的控制具有实时、原位、精确、快速的优势。该文综述了不同光敏基团的结构与功能,包括硝基苄基类、香豆素类、喹啉类、吲哚类、硝基二苯并呋喃类等,它们通过掩蔽神经递质、钙离子、蛋白质、缩氨酸、核苷酸、遗传物质等重要的生理活性物质来高选择性地调控不同的生物学过程。     

细菌L-天冬氨酸α-脱羧酶的分子机制及分子改造研究进展

L-天冬氨酸α-脱羧酶能够催化L-天冬氨酸生成β-丙氨酸,是泛酸代谢中的关键酶之一,对生物体中的能量代谢和脂肪代谢至关重要。细菌L-天冬氨酸α-脱羧酶属于丙酮酰依赖型的一类酶,具有特别的翻译后加工机制和底物失活作用,本文对L-天冬氨酸α-脱羧酶的分子机制和分子改造进行了综述,并对其在β-丙氨酸合成方面的应用进行了展望。     

A molecular beacon, bead-based assay for the detection of nucleic acids by flow cytometry

Molecular beacons are dual-labelled probes that are typically used in real-time PCR assays, but have also been conjugated with solid matrices for use in microarrays or biosensors. We have developed a fluid array system using microsphere-conjugated molecular beacons and the flow cytometer for the specific, multiplexed detection of unlabelled nucleic acids in solution. For this array system, molecular beacons were conjugated with microspheres using a biotin-streptavidin linkage. A bridged conjugation method using streptavidin increased the signal-to-noise ratio, allowing for further discrimination of target quantitation. Using beads of different sizes and molecular beacons in two fluorophore colours, synthetic nucleic acid control sequences were specifically detected for three respiratory pathogens, including the SARS coronavirus in proof-of-concept experiments. Considering that routine flow cytometers are able to detect up to four fluorescent channels, this novel assay may allow for the specific multiplex detection of a nucleic acid panel in a single tube.     

Hybridization kinetics and thermodynamics of molecular beacons

Molecular beacons are increasingly being used in many applications involving nucleic acid detection and quantification. The stem–loop structure of molecular beacons provides a competing reaction for probe–target hybridization that serves to increase probe specificity, which is particularly useful when single-base discrimination is desired. To fully realize the potential of molecular beacons, it is necessary to optimize their structure. Here we report a systematic study of the thermodynamic and kinetic parameters that describe the molecular beacon structure–function relationship. Both probe and stem lengths are shown to have a significant impact on the binding specificity and hybridization kinetic rates of molecular beacons. Specifically, molecular beacons with longer stem lengths have an improved ability to discriminate between targets over a broader range of temperatures. However, this is accompanied by a decrease in the rate of molecular beacon–target hybridization. Molecular beacons with longer probe lengths tend to have lower dissociation constants, increased kinetic rate constants, and decreased specificity. Molecular beacons with very short stems have a lower signal-to-background ratio than molecular beacons with longer stems. These features have significant implications for the design of molecular beacons for various applications.     

Shedding light on health and disease using molecular beacons.

The detection and identification of pathogens is often painstaking due to the low abundance of diseased cells in clinical samples. The genomic sequences of the pathogen can be amplified through methods such as the polymerase chain reaction and nucleic acid sequence-based amplification, but the nucleic acid targets are often lost among other unintended products of amplification. Novel nucleic acid probes known as molecular beacons have been developed allowing for the rapid and specific detection of genetic markers of a disease. Molecular beacons are hairpin-forming oligonucleotides labelled at one end with a quencher and at the other end with a fluorescent reporter dye. In the absence of target, the fluorescence is quenched. In the presence of target, the hairpin structure opens upon beacon/target hybridisation, resulting in the restoration of fluorescence. The ability to transduce target recognition into a fluorescence signal with high signal-to-background ratio, coupled with an improved specificity, has allowed molecular beacons to enjoy a wide range of biological and biomedical applications. Here, we describe the basic features of molecular beacons, review their applications in disease detection and diagnosis and discuss some of the issues and challenges of in vivo studies. The aim of this paper is to foster the development of new molecular beacon-based assays and to stimulate the application of this technology in laboratory and clinical studies of health and disease.     

Molecular beacons: nucleic acid hybridization and emerging applications.

Molecular beacons (MBs) are a novel class of nucleic acid probes that become fluorescent when bound to a complementary sequence. Because of this characteristic, coupled with the sequence specificity of nucleic acid hybridization and the sensitivity of fluorescence techniques, MBs are very useful probes for a variety of applications requiring the detection of DNA or RNA. We survey various applications of MBs, including the monitoring of DNA triplex formation, and describe recent developments in MB design that enhance their sensitivity.     

Molecular beacon sequence design algorithm

A method based on Web-based tools is presented to design optimally functioning molecular beacons. Molecular beacons, fluorogenic hybridization probes, are a powerful tool for the rapid and specific detection of a particular nucleic acid sequence. However, their synthesis costs can be considerable. Since molecular beacon performance is based on its sequence, it is imperative to rationally design an optimal sequence before synthesis. The algorithm presented here uses simple Microsoft Excel formulas and macros to rank candidate sequences. This analysis is carried out using mfold structural predictions along with other free Web-based tools. For smaller laboratories where molecular beacons are not the focus of research, the public domain algorithm described here may be usefully employed to aid in molecular beacon design.     

Biomolecular computation with molecular beacons for quantitative analysis of target nucleic acids

Molecular beacons are efficient and useful tools for quantitative detection of specific target nucleic acids. Thanks to their simple protocol, molecular beacons have great potential as substrates for biomolecular computing. Here we present a molecular beacon-based biomolecular computing method for quantitative detection and analysis of target nucleic acids. Whereas the conventional quantitative assays using fluorescent dyes have been designed for single target detection or multiplexed detection, the proposed method enables us not only to detect multiple targets but also to compute their quantitative information by weighted-sum of the targets. The detection and computation are performed on a molecular level simultaneously, and the outputs are detected as fluorescence signals. Experimental results show the feasibility and effectiveness of our weighted detection and linear combination method using molecular beacons. Our method can serve as a primitive operation of molecular pattern analysis, and we demonstrate successful binary classifications of molecular patterns made of synthetic oligonucleotide DNA molecules.     

Molecular beacons

This opinion covers the field of molecular beacons (MBs), in which nucleic acids are molecularly engineered to have unique functions for the investigation of biomolecules. Molecular beacons have been used in a variety of formats, and this review discusses four: first, in vitro RNA and DNA monitoring; second, biosensors and biochips based on MBs; third, real-time monitoring of genes and gene expression in living systems; and finally, the next generation of molecular beacons that will be highly useful for studies with proteins, molecular beacon aptamers. These unique applications have shown that MBs holds great potential in genomics and proteomics where real-time molecular recognition with high sensitivity and excellent specificity is critical.     

分子信标核酸检测技术研究进展

介绍了分子信标设计和分子信标核酸检测原理、技术特性和在基因突变大规模自动化检测中的应用. 分子信标是一种基于荧光共振能量转移现象设计的发卡型寡核苷酸探针,空间结构上呈茎环结构, 环序列是与靶核酸互补的探针,茎序列由与靶序列无关的互补序列构成,茎的一端连上荧光分子,另一端连上淬灭分子.通过空间结构改变决定分子信标发射荧光特性,从而对核酸进行定量检测. 分子信标技术具有操作简单、敏感、特异、可对核酸进行液相实时检测和对活体内核酸动态进行检测等特点,已应用于HIV辅助受体基因等基因突变的大规模自动化检测,是一种新型核酸定量检测技术.     

Tripartite molecular beacons

Molecular beacons (MBs) are hairpin-like fluorescent DNA probes that have single-mismatch detection capability. Although they are extremely useful for many solution-based nucleic acid detections, MBs are expensive probes for applications that require the use of a large number of different DNA probes due to the high cost and tedious procedures associated with probe synthesis and purification. In addition, since both ends of MB probes are covalently modified with chromophores, they do not offer the flexibility for fluorophore change and the capability for surface immobilization through free DNA ends. In this report, we describe an alternative form of MB, denoted tripartite molecular beacon (TMB), that may help overcome these problems. A TMB uses an unmodified oligodeoxyribonucleotide that forms a MB-like structure with two universal single-stranded arms to bring on a universal pair of oligodeoxyribonucleotides modified separately with a fluorophore and a quencher. We found that TMBs are as effective as standard MBs in signaling the presence of matching nucleic acid targets and in precisely discriminating targets that differ by a single nucleotide. TMBs have the necessary flexibility that may make MBs more affordable for various nucleic acid detection applications.     

Exciton interaction in molecular beacons: a sensitive sensor for short range modifications of the nucleic acid structure

Molecular beacons are hairpin-shaped, single-stranded oligonucleotides constituting sensitive fluorescent DNA probes widely used to report the presence of specific nucleic acids. In its closed form the stem of the hairpin holds the fluorophore covalently attached to one end, close to the quencher, which is covalently attached to the other end. Here we report that in the closed form the fluorophore and the quencher form a ground state intramolecular heterodimer whose spectral properties can be described by exciton theory. Formation of the heterodimers was found to be poorly sensitive to the stem sequence, the respective positions of the dyes and the nature of the nucleic acid (DNA or RNA). The heterodimer allows strong coupling between the transition dipoles of the two chromophores, leading to dramatic changes in the absorption spectrum that are not compatible with a Förster-type fluorescence resonance energy transfer (FRET) mechanism. The excitonic heterodimer and its associated absorption spectrum are extremely sensitive to the orientation of and distance between the dyes. Accordingly, the application of molecular beacons can be extended to monitoring short range modifications of the stem structure. Moreover, the excitonic interaction was also found to operate for doubly end-labeled duplexes.