多元光学蛋白质芯片研究取得重要进展

蛋白质芯片技术是一种新型蛋白质分析技术,具有集成、并行、快速和自动化分析的优势。多元光学蛋白质芯片传感器,仅需微量生理或生物采样,即可以同时检测、识别和纯化不同的生物分子和研究分子间的相互作用。无需标记,可以直接测量像血浆、细胞裂解液等生理样品。     

单分子层次上相互作用自动检测

任何生命过程都与分子间相互作用有关。这些相互作用决定了生物分子间的＂交流＂方式,组成了生物过程的基本语言。Müller教授研究组发展了一种全自动＂机器人＂（一种全自动原子力显微镜）,可以通过检测细胞上的＂分子机器＂分析分子间相互作用。为了实现这样的目标,该仪器需要将不同的空间尺度联系起来：宏观尺度的悬臂利用其微观尺度的针尖与纳米尺度的蛋白质相接触,进而在亚纳米的尺度上定位与检测分子间相互作用。这项技术能够帮助人们以亚纳米尺度的分辨率定位细胞内分子机器的相互作用位置,并且观察分子间相互作用如何驱动这些分子机器行使功能。在药物筛选研究领域,该技术可以被用来检测配体以及抑制剂与蛋白质结合的位点和强度,还可以检测受体的不同功能状态。     

邻近标记在蛋白质组学中的发展及应用

人体内各种复杂的生命活动离不开蛋白质之间的相互作用。这种相互作用具有瞬时性和结合力弱等特点,并受到多种动态调节,特别是蛋白质翻译后修饰（post-translation modifications, PTM）。传统的亲和质谱检测方法存在蛋白纯化的局限性,在高效检测到动态变化方面存在不足。邻近标记是一种能够给与靶蛋白质瞬时靠近,或者互作（邻近）的蛋白质加上生物素的技术,它与质谱检测技术的联合使用能检测细胞过程中弱的、瞬时的蛋白质相互作用,有效解决上述问题。本文综述了基于生物素的邻近标记方法的发展现状,从依赖于融合序列的生物素标记开始,依次介绍有关生物素连接酶、过氧化物酶及其进化后的2代标记方法等经典生物素标记的方法和原理,比较各个方法间的差异和优缺点;也列举了一些近年来新出现的标记方法,如将生物素连接酶进行拆分、鉴定蛋白质在不同复合物中功能的方法、抗体靶向的标记方法,以及其他来源的生物素连接酶突变体,例如枯草芽孢杆菌（Bacillus subtilis）的C端氨基酸突变的生物素连接酶,能够应用在苍蝇和蠕虫中的生物素连接酶突变体。本文对这些方法进行归纳总结,旨在为初步接触该领域的科研工作者提供参考,同时也希望能够提供一些新的思路,推动蛋白质相互作用组学的发展。     

邻近标记在蛋白质组学中的发展及应用

人体内各种复杂的生命活动离不开蛋白质之间的相互作用。这种相互作用具有瞬时性和结合力弱等特点,并受到多种动态调节,特别是蛋白质翻译后修饰（post-translation modifications, PTM）。传统的亲和质谱检测方法存在蛋白纯化的局限性,在高效检测到动态变化方面存在不足。邻近标记是一种能够给与靶蛋白质瞬时靠近,或者互作（邻近）的蛋白质加上生物素的技术,它与质谱检测技术的联合使用能检测细胞过程中弱的、瞬时的蛋白质相互作用,有效解决上述问题。本文综述了基于生物素的邻近标记方法的发展现状,从依赖于融合序列的生物素标记开始,依次介绍有关生物素连接酶、过氧化物酶及其进化后的2代标记方法等经典生物素标记的方法和原理,比较各个方法间的差异和优缺点;也列举了一些近年来新出现的标记方法,如将生物素连接酶进行拆分、鉴定蛋白质在不同复合物中功能的方法、抗体靶向的标记方法,以及其他来源的生物素连接酶突变体,例如枯草芽孢杆菌（Bacillus subtilis）的C端氨基酸突变的生物素连接酶,能够应用在苍蝇和蠕虫中的生物素连接酶突变体。本文对这些方法进行归纳总结,旨在为初步接触该领域的科研工作者提供参考,同时也希望能够提供一些新的思路,推动蛋白质相互作用组学的发展。     

光镊探索DNA凝聚过程

自从20年前光镊技术被Ashkin、Chu及其同事发明[1],该技术已被应用于各种生物学研究并由此获得丰富信息.例如应力下生物高分子的行为[2],DNA连接酶如何译码或如何消化DNA[3～6],动力蛋白如何沿分子轨道移动[7～8],以及RNA和蛋白质分子如何折叠/展开[9～11].通过对单个分子的操纵,光镊技术可用于探索这些生物系统在分子层面的工作模式.在本期222页,Case等描述了该技术的另一精巧应用.揭示了凝聚子蛋白质如何产生紧凑形式的DNA[12].     

纳米生物条形码技术在分子诊断中的应用

纳米生物条形码技术是一种新型的分子诊断技术,在核酸和蛋白质检测领域已经分别达到和超过现今的检测灵敏度.该技术采用纳米颗粒修饰的核酸或抗体对靶分子进行识别,并利用磁场将其分离,操作简单,不依赖酶反应,具有广泛的应用前景.该技术在病毒DNA、癌症的指标蛋白质等方面的应用已有文献报道,并有望成为一种常规的分子诊断工具.     

生物信息学方法预测蛋白质相互作用网络中的功能模块

蛋白质相互作用是大多数生命过程的基础。随着高通量实验技术和计算机预测方法的发展,在各种生物中已获得了数目十分庞大的蛋白质相互作用数据,如何从中提取出具有生物学意义的数据是一项艰巨的挑战。从蛋白质相互作用数据出发获得相互作用网络进而预测出其中的功能模块,对于蛋白质功能预测、揭示各种生化反应过程的分子机理都有着极大的帮助。我们分类概括了用生物信息学预测蛋白质相互作用功能模块的方法,以及对这些方法的评价,并介绍了蛋白质相互作用网络比较的一些方法。     

双杂交和质谱技术在蛋白质-蛋白质相互作用研究中的应用

基因的功能是由蛋白质来执行的,而蛋白质要通过与其他生物分子相互作用来完成其各种生物功能。因此,如果能够快速做出蛋白质在不同时间、空间和不同环境中的相互作用图谱,就会帮助我们了解这些蛋白质的功能,进而了解许多生命活动的机制。目前,用于大规模研究蛋白质间相互作用的方法主要有酵母双杂交系统及其衍生系统、亲和纯化与质谱分析联用技术,前者用于研究蛋白分子间的两两相互作用,后者用于研究蛋白质复合物间的相互作用。本文主要阐述了酵母双杂交、细菌双杂交、哺乳动物细胞双杂交、亲和纯化与质谱联用技术在大规模蛋白质相互作用研究中的应用。     

见微知著:单分子力谱技术窥探蛋白质分子动态功能北大核心CSCD

蛋白质的动态功能调控并决定着细胞的生理和病理过程。其不仅受到蛋白质本身生物化学特性的影响,还受到生物体内复杂的生物力学微环境的动态调控。这些生物力因素主要通过耦联生物化学特性来改变蛋白质的动态相互作用、构象变化以及后续的信号传导。近些年来,单分子力谱检测技术突破了传统生物化学技术的限制,在单分子水平有效地研究生物力学——化学耦联调控下的蛋白质动态功能。本文详细介绍了4种代表性的单分子力谱检测技术(原子力显微镜、光镊、生物膜力学探针以及磁镊),着重介绍这些技术在蛋白质动态功能研究方面的典型应用,主要包括蛋白质动态相互作用,蛋白质动态构象变化以及信号传导等。同时,本文还介绍了几种常用的基于上述单分子检测技术的单分子力谱检测方法,主要用于定量检测蛋白质相互作用、构象变化等生物化学过程的分子动力学参数。最后,本文还简要讨论了单分子力谱检测技术的未来发展方向,特别是如何与其他研究手段的有机整合,更全面地研究蛋白质的动态功能。我们希望该综述能够给更多的生物化学家带来新的概念和工具,帮助更全面地研究蛋白质的动态特性。     

大规模蛋白质相互作用组实验技术及其应用

大规模蛋白质相互作用研究的主要实验技术包括酵母双杂交技术、串联亲和纯化技术和蛋白质芯片技术,随着这些技术的不断发展和完善,科学家们在模式生物、哺乳动物、病原微生物中展开了大规模的蛋白质相互作用组研究,并进行了药物研发方面的研究,绘制了多种生物的蛋白质相互作用连锁图,揭示了多种蛋白质的新功能,为全面研究蛋白质（群）的分子作用机制、药物研发和疾病的临床预防与治疗等提供了崭新的线索。     

Fluorescent oligonucleotide ligation technology for identification of ras oncogene mutations

A mutation detection strategy based on multiplex PCR followed by multiplex allele-specific oligonucleotide probe ligation was developed to detect single nucleotide substitutions in ras oncogenes, a common genetic abnormality in many human cancers. Mutation-specific probes are synthesized for each possible single-base, nonsilent mutation in codons 12, 13, and 61 of H-, K-, and N-ras oncogenes. Mutations are identified by competitive oligonucleotide probe ligation to detect normal and /or mutant genotypes in one reaction. Three probes (one common and two allelic probes) are needed for analysis of each mutation. Probes hybridized to target ras oncogene DNA are joined by a thermostable ligase if there are no mismatches at their junctions; temperature cycling results in a linear increase in product. Common probes are labeled with fluorochromes, and allelic probes each have different lengths. Ligation products are analyzed by denaturing polyacrylamide gel electrophoresis on a fluorescent DNA sequencer. We have applied this technology to identify ras mutations in pancreatic cancers and lung cancers and in patients with myelodysplastic syndromes and leukemias.     

Universal oligonucleotide microarray for sub-typing of Influenza A virus

A universal microchip was developed for genotyping Influenza A viruses. It contains two sets of oligonucleotide probes allowing viruses to be classified by the subtypes of hemagglutinin (H1-H13, H15, H16) and neuraminidase (N1-N9). Additional sets of probes are used to detect H1N1 swine influenza viruses. Selection of probes was done in two steps. Initially, amino acid sequences specific to each subtype were identified, and then the most specific and representative oligonucleotide probes were selected. Overall, between 19 and 24 probes were used to identify each subtype of hemagglutinin (HA) and neuraminidase (NA). Genotyping included preparation of fluorescently labeled PCR amplicons of influenza virus cDNA and their hybridization to microarrays of specific oligonucleotide probes. Out of 40 samples tested, 36 unambiguously identified HA and NA subtypes of Influenza A virus.     

probeBase: an online resource for rRNA-targeted oligonucleotide probes

Ribosomal RNA-(rRNA)-targeted oligonucleotide probes are widely used for culture-independent identification of microorganisms in environmental and clinical samples. ProbeBase is a comprehensive database containing more than 700 published rRNA-targeted oligonucleotide probe sequences (status August 2002) with supporting bibliographic and biological annotation that can be accessed through the internet at http://www.probebase.net. Each oligonucleotide probe entry contains information on target organisms, target molecule (small- or large-subunit rRNA) and position, G+C content, predicted melting temperature, molecular weight, necessity of competitor probes, and the reference that originally described the oligonucleotide probe, including a link to the respective abstract at PubMed. In addition, probes successfully used for fluorescence in situ hybridization (FISH) are highlighted and the recommended hybridization conditions are listed. ProbeBase also offers difference alignments for 16S rRNA-targeted probes by using the probe match tool of the ARB software and the latest small-subunit rRNA ARB database (release June 2002). The option to directly submit probe sequences to the probe match tool of the Ribosomal Database Project II (RDP-II) further allows one to extract supplementary information on probe specificities. The two main features of probeBase, 'search probeBase' and 'find probe set', help researchers to find suitable, published oligonucleotide probes for microorganisms of interest or for rRNA gene sequences submitted by the user. Furthermore, the 'search target site' option provides guidance for the development of new FISH probes.     

Comparisons of substitution, insertion and deletion probes for resequencing and mutational analysis using oligonucleotide microarrays

Although oligonucleotide probes complementary to single nucleotide substitutions are commonly used in microarray-based screens for genetic variation, little is known about the hybridization properties of probes complementary to small insertions and deletions. It is necessary to define the hybridization properties of these latter probes in order to improve the specificity and sensitivity of oligonucleotide microarray-based mutational analysis of disease-related genes. Here, we compare and contrast the hybridization properties of oligonucleotide microarrays consisting of 25mer probes complementary to all possible single nucleotide substitutions and insertions, and one and two base deletions in the 9168 bp coding region of the ATM (ataxia telangiectasia mutated) gene. Over 68 different dye-labeled single-stranded nucleic acid targets representing all ATM coding exons were applied to these microarrays. We assess hybridization specificity by comparing the relative hybridization signals from probes perfectly matched to ATM sequences to those containing mismatches. Probes complementary to two base substitutions displayed the highest average specificity followed by those complementary to single base substitutions, single base deletions and single base insertions. In all the cases, hybridization specificity was strongly influenced by sequence context and possible intra- and intermolecular probe and/or target structure. Furthermore, single nucleotide substitution probes displayed the most consistent hybridization specificity data followed by single base deletions, two base deletions and single nucleotide insertions. Overall, these studies provide valuable empirical data that can be used to more accurately model the hybridization properties of insertion and deletion probes and improve the design and interpretation of oligonucleotide microarray-based resequencing and mutational analysis.     

Determinants of sensitivity and specificity in spotted DNA microarrays with unmodified oligonucleotides

DNA microarrays with unmodified oligonucleotides are a cost-effective alternative to cDNA microarrays. This study examined how purity, length, homology and GC content of the oligonucleotide probes influence the sensitivity and specificity of the method using cyanobacterial genes. Oligonucleotide purification by high pressure liquid chromatography was omitted without significant reduction in hybridization sensitivity. For two of three genes tested, a reduction in oligonucleotide length did not reduce hybridization sensitivity, and maximum sensitivity was achieved with probes that were 45 nt long. Oligonucleotide probes with 相似文献   

Fluorescent pteridine nucleoside analogs

Pteridine nucleoside analog probes are highly fluorescent and offer different approaches to monitor subtle DNA interactions with other molecules. Similarities in structure and size to native nucleosides make it possible to incorporate these probes into oligonucleotides through the standard deoxyribose linkage. These probes are formulated as phosphoramidites and incorporated into oligonucleotides using automated DNA synthesis. Their position within the oligonucleotide renders them exquisitely sensitive to changes in structure as the oligonucleotide meets and reacts with other molecules. Changes are measured through fluorescence intensity, anisotropy, lifetimes, spectral shifts, and energy transfer. The fluorescence properties of pteridine nucleoside analogs as monomers and incorporated into single and double stranded oligonucleotides are reviewed. The two guanosine analogs, 3MI and 6MI, and two adenosine analogs, 6MAP and DMAP, are reviewed in detail along with applications utilizing them.     

Real-time genotyping with oligonucleotide probes containing locked nucleic acids

Oligonucleotide probes containing locked nucleic acid (LNA) hybridize to complementary single-stranded target DNA sequences with an increased affinity compared to oligonucleotide DNA probes. As a consequence of the incorporation of LNA residues into the oligonucleotide sequence, the melting temperature of the oligonucleotide increases considerably, thus allowing the successful use of shorter LNA probes as allele-specific tools in genotyping assays. In this article, we report the use of probes containing LNA residues for the development of qualitative fluorescent multiplex assays for the detection of single nucleotide polymorphisms (SNPs) in real-time polymerase chain reaction using the 5'-nuclease detection assay. We developed two applications that show the improved specificity of LNA probes in assays for allelic discrimination. The first application is a four-color 5'-nuclease assay for the detection of SNPs for two of the most common genetic factors involved in thrombotic risk, factor V Leiden and prothrombin G20210A. The second application is a two-color assay for the specific detection of the A-to-T tranversion in codon 6 of the beta-globin gene, responsible for sickle cell anemia. Both real-time genotyping assays were evaluated by comparing the performance of our method to that of a reference method and in both cases, we found a 100% concordance. This approach will be useful for research and molecular diagnostic laboratories in situations in which the specificity provided by oligonucleotide DNA probes is insufficient to discriminate between two DNA sequences that differ by only one nucleotide.