Mass spectrometry of RNA: linking the genome to the proteome.

Ribonucleic acids (RNAs) are continuing to attract increased attention as they are found to play pivotal roles in biological systems. Just as genomics and proteomics have been enabled by the development of effective analytical techniques and instrumentation, the large-scale analysis of non-protein coding (nc)RNAs will benefit as new analytical methodologies, such as mass spectrometry (MS), are developed for their analysis. Mass spectrometry offers a number of advantages for RNA analysis arising from its ability to provide mass and sequence information starting with limited amounts of sample. This review will highlight recent developments in the field of MS that enable the characterization of RNA modification status, RNA tertiary structures, and ncRNA expression levels. These developments will also be placed in perspective of how MS of RNAs can help elucidate the link between the genome and proteome.     

Mass spectrometry of nucleic acids in molecular medicine

A stable streamlining trend in the field of medical diagnostics by practical adoption of high-tech and knowledge-intensive analytical systems providing for molecular level studies has appeared during the last few decades. An illustrative example of such technologies is mass spectrometry methods for analyzing biomolecules. This review is intended to brief the potential of the state-of-the-art inventory of spectrometry equipment and illustrate the application of mass spectrometry of nucleic acids (DNA and RNA) for solving practical problems related to the analysis of human genomic DNA and clinically significant microorganisms of bacterial and viral natures.     

Mass spectrometry-based proteomics for systems biology

Mass spectrometry (MS)-based proteomics has significantly contributed to the development of systems biology, a new paradigm for the life sciences in which biological processes are addressed in terms of dynamic networks of interacting molecules. Because of its advanced analytical capabilities, MS-based proteomics has been used extensively to identify the components of biological systems, and it is the method of choice to consistently quantify the effects of network perturbation in time and space. Herein, we review recent contributions of MS to systems biology and discuss several examples that illustrate the importance of mass spectrometry to elucidate the components and interactions of molecular networks.     

Biological signalling activity measurements using mass spectrometry

MS (mass spectrometry) techniques are rapidly evolving to high levels of performance and robustness. This is allowing the application of these methods to the interrogation of signalling networks with unprecedented depth and accuracy. In the present review we discuss how MS-based multiplex quantification of kinase activities and phosphoproteomics provide complementary means to assess biological signalling activity. In addition, we discuss how a wider application of these analytical concepts to quantify kinase signalling will result in a more comprehensive understanding of normal and disease biology at the system level.     

DNA mimics based on pyrrolidine and hydroxyproline

In recent years, a great number of analogues and mimics of nucleic acids have been developed with the aim of improving the physicochemical and biological properties of native oligonucleotides, in particular, to increase their affinity for nucleic acids, selectivity of action, and biological stability. This review summarizes the data on the synthesis and properties of DNA mimics, the analogues of peptide nucleic acids, which are the derivatives of pyrrolidine and hydroxyproline. Some physicochemical and biological properties of negatively charged mimics of this type are considered, which contain phosphonate residues in the back-bone and exhibit a high affinity for DNA and RNA, the selectivity of binding to nucleic acids, and stability in various biological systems. Examples of using these mimics as tools in molecular biology studies, in particular, functional genomics, are given. The prospects for their application in diagnosis and medicine are discussed.     

NMR studies of dynamics in RNA and DNA by 13C relaxation

RNA and DNA molecules experience motions on a wide range of time scales, ranging from rapid localized motions to much slower collective motions of entire helical domains. The many functions of RNA in biology very often require this molecule to change its conformation in response to biological signals in the form of small molecules, proteins or other nucleic acids, whereas local motions in DNA may facilitate protein recognition and allow enzymes acting on DNA to access functional groups on the bases that would otherwise be buried in Watson-Crick base pairs. Although these statements make a compelling case to study the sequence dependent dynamics in nucleic acids, there are few residue-specific studies of nucleic acid dynamics. Fortunately, NMR studies of dynamics of nucleic acids and nucleic acids-protein complexes are gaining increased attention. The aim of this review is to provide an update of the recent progress in studies of nucleic acid dynamics by NMR based on the application of solution relaxation techniques.     

合成生物学及其在生物技术中的应用进展

合成生物学是一门21世纪生物学的新兴学科,它着眼生物科学与工程科学的结合,把生物系统当作工程系统"从下往上"进行处理,由"单元"(unit)到"部件"(device)再到"系统"(system)来设计,修改和组装细胞构件及生物系统.合成生物学是分子和细胞生物学、进化系统学、生物化学、信息学、数学、计算机和工程等多学科交叉的产物.目前研究应用包括两个主要方面:一是通过对现有的、天然存在的生物系统进行重新设计和改造,修改已存在的生物系统,使该系统增添新的功能.二是通过设计和构建新的生物零件、组件和系统,创造自然界中尚不存在的人工生命系统.合成生物学作为一门建立在基因组方法之上的学科,主要强调对创造人工生命形态的计算生物学与实验生物学的协同整合.必须强调的是,用来构建生命系统新结构、产生新功能所使用的组件单元既可以是基因、核酸等生物组件,也可以是化学的、机械的和物理的元件.本文跟踪合成生物学研究及应用,对其在DNA水平编程、分子修饰、代谢途径、调控网络和工业生物技术等方面的进展进行综述.     

Nobel Prize 2002 for chemistry: mass spectrometry and nuclear magnetic resonance

The Nobel Prize in Chemistry for 2002 has been awarded to two powerful spectroscopic methodologies through three valorous scientists, John Fenn and Koichi Tanaka, for mass spectrometry and Kurt Wüthrich for nuclear magnetic resonance. These techniques were previously known for their intensive use in chemical analysis. They are now developed at the chemistry/biology interface. Two new methods of soft ionization in mass spectrometry and a strategy of sequential assignment of nuclear magnetic resonance signals of biopolymers now allow the use of these powerful and complementary methodologies for the structural analysis of biological macromolecules, proteins, nucleic acids (DNA, RNA) and polysaccharides. Through the elucidation of their planar and three-dimensional structures and of the molecular mechanisms that govern their interactions, these techniques now may afford precious clues for understanding the molecular mechanisms of life.     

Surface-assisted laser desorption/ionization mass spectrometry

Laser desorption/ionization mass spectrometry (MS) is rapidly growing in popularity as an analytical characterization method in several fields. The technique shot to prominence using matrix-assisted desorption/ionization for large biomolecules (>700 Da), such as proteins, peptides and nucleic acids. However, because the matrix, which consists of small organic molecules, is also ionized, the technique is of limited use in the low-molecular-mass range (<700 Da). Recent advances in surface science have facilitated the development of matrix-free laser desorption/ionization MS approaches, which are referred to here as surface-assisted laser desorption/ionization (SALDI) MS. In contrast to traditional matrix-assisted techniques, the materials used for SALDI-MS are not ionized, which expands the usefulness of this technique to small-molecule analyses. This review discusses the current status of SALDI-MS as a standard analytical technique, with an emphasis on potential applications in proteomics.     

Surface-assisted laser desorption/ionization mass spectrometry

Laser desorption/ionization mass spectrometry (MS) is rapidly growing in popularity as an analytical characterization method in several fields. The technique shot to prominence using matrix-assisted desorption/ionization for large biomolecules (>700 Da), such as proteins, peptides and nucleic acids. However, because the matrix, which consists of small organic molecules, is also ionized, the technique is of limited use in the low-molecular-mass range (<700 Da). Recent advances in surface science have facilitated the development of matrix-free laser desorption/ionization MS approaches, which are referred to here as surface-assisted laser desorption/ionization (SALDI) MS. In contrast to traditional matrix-assisted techniques, the materials used for SALDI-MS are not ionized, which expands the usefulness of this technique to small-molecule analyses. This review discusses the current status of SALDI-MS as a standard analytical technique, with an emphasis on potential applications in proteomics.     

Targeted proteome investigation via selected reaction monitoring mass spectrometry

Due to the enormous complexity of proteomes which constitute the entirety of protein species expressed by a certain cell or tissue, proteome-wide studies performed in discovery mode are still limited in their ability to reproducibly identify and quantify all proteins present in complex biological samples. Therefore, the targeted analysis of informative subsets of the proteome has been beneficial to generate reproducible data sets across multiple samples. Here we review the repertoire of antibody- and mass spectrometry (MS) -based analytical tools which is currently available for the directed analysis of predefined sets of proteins. The topics of emphasis for this review are Selected Reaction Monitoring (SRM) mass spectrometry, emerging tools to control error rates in targeted proteomic experiments, and some representative examples of applications. The ability to cost- and time-efficiently generate specific and quantitative assays for large numbers of proteins and posttranslational modifications has the potential to greatly expand the range of targeted proteomic coverage in biological studies. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.     

生物质谱技术及其应用

质谱是带电粒子按质荷比大小顺序排列的图谱,最初主要用来测定元素或同位素的原子量,随着科学的发展及高性能质谱仪器的出现,质谱被越来越多地应用生命科学研究的许多领域,以其质辅助激光解吸附飞行时间质谱和电喷雾质谱为代表的现代生物质谱技术,为蛋白质等生物大分子的研究提供了必要的技术手段。本文在简介近年来比较常用的几种生物质谱技术的基础上,概述了生物质谱技术在蛋白质,核酸研究及检测分析等几个方面的初步应用。     

The essence of DNA sample preparation for MALDI mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has become an important analytical technique in nucleic acid research. MALDI is used for quality control of oligonucleotides as well as for analyzing DNA markers. Sample preparation of nucleic acids is crucial for obtaining high-quality mass spectra. Sample purity, solvent content, suitable matrices, and substrate surfaces, as well as laboratory conditions affect spectra quality. This review presents essential information with regard to sample preparation, DNA modification chemistry, and DNA purification, along with a discussion of instrumental advances, which facilitate and extend the applicability of MALDI in genomics.     

Smart Nanocarriers for the Delivery of Nucleic Acid-Based Therapeutics: A Comprehensive Review

Nucleic acid-based therapies are promising therapeutics for the treatment of several systemic disorders, and they offer an exciting opportunity to address emerging biological challenges. The scope of nucleic acid-based therapeutics in the treatment of multiple disease states including cancers has been widened by recent progress in Ribonucleic acids (RNA) biology. However, cascades of systemic and intracellular barriers, including rapid degradation, renal clearance, and poor cellular uptake, hinder the clinical effectiveness of nucleic acid-based therapies. These barriers can be circumvented by utilizing advanced smart nanocarriers that efficiently deliver and release the encapsulated nucleic acids into the target tissues. This review describes the current status of clinical trials on nucleic acid-based therapeutics and highlights representative examples that provide an overview on the current and emerging trends in nucleic acid-based therapies. A better understanding of the design of advanced nanocarriers is essential to promote the translation of therapeutic nucleic acids into a clinical reality.     

The essence of DNA sample preparation for MALDI mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has become an important analytical technique in nucleic acid research. MALDI is used for quality control of oligonucleotides as well as for analyzing DNA markers. Sample preparation of nucleic acids is crucial for obtaining high-quality mass spectra. Sample purity, solvent content, suitable matrices, and substrate surfaces, as well as laboratory conditions affect spectra quality. This review presents essential information with regard to sample preparation, DNA modification chemistry, and DNA purification, along with a discussion of instrumental advances, which facilitate and extend the applicability of MALDI in genomics.     

An ESI-MS method for characterization of native and modified oligonucleotides used for RNA interference and other biological applications

RNA interference (RNAi) has become a powerful tool for investigating gene function, and, in addition, shows potential for the development of therapeutic agents. RNAi can be triggered in a variety of eukaryotic cells using small interfering RNA (siRNA), their double-stranded precursors (double-stranded RNA) and short hairpin precursors (shRNA). Here, we describe a protocol for analyzing these RNAs and their modifications using electrospray ionization mass spectrometry (ESI-MS). This protocol involves the desalting of nucleic acids using ammonium acetate precipitation, followed by characterization using ESI-MS. This protocol has been chiefly used for analyzing siRNAs and their chemical modifications, but it has also been used and can be applied to the analysis of a wide range of native and modified oligonucleotides. This protocol provides accurate information on molecular weight for a range of nucleic acids and can be completed in less than a day.