Selenium Derivatization of Nucleic Acids for X‐Ray Crystal‐Structure and Function Studies

It is estimated that over two thirds of all new crystal structures of proteins are determined via the protein selenium derivatization (selenomethionine (Se‐Met) strategy). This selenium derivatization strategy via MAD (multi‐wavelength anomalous dispersion) phasing has revolutionized protein X‐ray crystallography. Through our pioneer research, similarly, Se has also been successfully incorporated into nucleic acids to facilitate the X‐ray crystal‐structure and function studies of nucleic acids. Currently, Se has been stably introduced into nucleic acids by replacing nucleotide O‐atom at the positions 2′, 4′, 5′, and in nucleobases and non‐bridging phosphates. The Se derivatization of nucleic acids can be achieved through solid‐phase chemical synthesis and enzymatic methods, and the Se‐derivatized nucleic acids (SeNA) can be easily purified by HPLC, FPLC, and gel electrophoresis to obtain high purity. It has also been demonstrated that the Se derivatization of nucleic acids facilitates the phase determination via MAD phasing without significant perturbation. A growing number of structures of DNAs, RNAs, and protein–nucleic acid complexes have been determined by the Se derivatization and MAD phasing. Furthermore, it was observed that the Se derivatization can facilitate crystallization, especially when it is introduced to the 2′‐position. In addition, this novel derivatization strategy has many advantages over the conventional halogen derivatization, such as more choices of the modification sites via the atom‐specific substitution of the nucleotide O‐atom, better stability under X‐ray radiation, and structure isomorphism. Therefore, our Se‐derivatization strategy has great potentials to provide rational solutions for both phase determination and high‐quality crystal growth in nucleic‐acid crystallography. Moreover, the Se derivatization generates the nucleic acids with many new properties and creates a new paradigm of nucleic acids. This review summarizes the recent developments of the atomic site‐specific Se derivatization of nucleic acids for structure determination and function study. Several applications of this Se‐derivatization strategy in nucleic acid and protein research are also described in this review.     

核酸试纸条检测方法研究进展*

凝胶电泳、实时荧光PCR等常规核酸检测方法存在操作繁琐、设备昂贵、反应时间长等局限性。随着核酸检测市场规模的大幅提升,常规检测方法已无法满足临床诊断、检验检疫的需求。核酸试纸条(nucleic acid detection strip,NADS)是一种新兴的核酸检测方法,具有灵敏度高、操作便捷、结果可视化、成本低且耗时短等优势,在基础研究与临床诊断等领域受到广泛关注。综述近年来NADS的检测方法及研究进展,系统总结该技术的原理、应用及临床潜在转化价值,以期为NADS的进一步开发、利用提供借鉴。     

核酸疫苗的研究及应用进展

核酸疫苗(DNA疫苗)是20世纪90年代发展起来的一种新型疫苗。核酸疫苗不仅可引起体液免疫反应,而且能诱导高水平的细胞免疫应答,尤其是细胞毒T淋巴细胞(CTL)反应,被认为在病毒、细菌、寄生虫等病原体感染的防治中具有更大的优势。核酸疫苗既可作为病毒、细菌或寄生虫的预防疫苗,也可作为非感染性疾病如肿瘤病的治疗用疫苗。     

Locked Nucleic Acids: Promising Nucleic Acid Analogs for Therapeutic Applications

Locked Nucleic Acid (LNA) is a unique nucleic‐acid modification possessing very high binding affinity and excellent specificity toward complementary RNA or DNA oligonucleotides. The remarkable properties exhibited by LNA oligonucleotides have been employed in different nucleic acid‐based therapeutic strategies both in vitro and in vivo. Herein, we highlight the applications of LNA nucleotides for controlling gene expression.     

静电泳动核酸电镜技术

本文提出了一种新的核酸电镜技术,即静电泳动核酸电镜技术。这种方法较常规的核酸展层技术操作简便,省工省时,节省试剂,效果令人满意。     

食源性病毒核酸恒温检测技术研究进展

食源性病毒已成为全球引发食品安全事件的重要病原,对新型检测技术的不断发展提出了严峻的挑战.早期PCR技术在病原检测领域中的应用,推动了对食源性病毒的全面认识.近年来核酸恒温检测技术发展迅速,包括环介导等温扩增技术、重组酶聚合酶扩增技术、核酸序列依赖性扩增技术、链置换扩增技术、滚环扩增技术等,在抗复杂基质干扰、装备要求低...     

Magnetic particles for the separation and purification of nucleic acids

Nucleic acid separation is an increasingly important tool for molecular biology. Before modern technologies could be used, nucleic acid separation had been a time- and work-consuming process based on several extraction and centrifugation steps, often limited by small yields and low purities of the separation products, and not suited for automation and up-scaling. During the last few years, specifically functionalised magnetic particles were developed. Together with an appropriate buffer system, they allow for the quick and efficient purification directly after their extraction from crude cell extracts. Centrifugation steps were avoided. In addition, the new approach provided for an easy automation of the entire process and the isolation of nucleic acids from larger sample volumes. This review describes traditional methods and methods based on magnetic particles for nucleic acid purification. The synthesis of a variety of magnetic particles is presented in more detail. Various suppliers of magnetic particles for nucleic acid separation as well as suppliers offering particle-based kits for a variety of different sample materials are listed. Furthermore, commercially available manual magnetic separators and automated systems for magnetic particle handling and liquid handling are mentioned.     

Nucleic acid structure and sequence probing using fluorescent base analogue tC(O)

The fluorescent cytosine analog tC(O) is on average the brightest probe of its kind and, moreover, it introduces minimal perturbations to the normal secondary structure of DNA. Here several ways of how tC(O), with an advantage, can be used as a local fluorescent probe in nucleic acid systems are presented. Most importantly, we show that tC(O) is an excellent probe for the detection of individual melting processes of complex nucleic acid structures containing a large number of separate secondary structure motifs. Since conventional UV-melting investigations merely monitor the global melting process of the whole nucleic acid structure, e.g. multi-hairpin systems in RNA/DNA, and thus is incapable of estimating individual melting transitions of such systems, tC(O) represents a new method of characterization. Furthermore, we find that tC(O) may be used to detect bulges and loops in nucleic acids as well as to distinguish a matched base-pair from several of the mismatched.     

双歧杆菌分泌RNA的实验

目的：通过双歧杆菌对数生长期培养液中出现核酸的性质的研究探索双歧杆菌作用于机体的分子机制。方法：采用液体培养双歧杆菌方法;提取对数生长的双歧杆菌培养发酵液中的总核酸;将纯化后的核酸用RNA降解酶降解,电泳观察核酸条带电泳结果。结果：双歧杆菌对数生长期培养发酵液中仅存在RNA。结论：双歧杆菌对数生长期可能分泌100bp RNA。     

Crystallographic Studies of Chemically Modified Nucleic Acids: A Backward Glance

Chemically modified nucleic acids (CNAs) are widely explored as antisense oligonucleotide or small interfering RNA (siRNA) candidates for therapeutic applications. CNAs are also of interest in diagnostics, high‐throughput genomics and target validation, nanotechnology and as model systems in investigations directed at a better understanding of the etiology of nucleic acid structure, as well as the physicochemical and pairing properties of DNA and RNA, and for probing protein–nucleic acid interactions. In this article, we review research conducted in our laboratory over the past two decades with a focus on crystal‐structure analyses of CNAs and artificial pairing systems. We highlight key insights into issues ranging from conformational distortions as a consequence of modification to the modulation of pairing strength, and RNA affinity by stereoelectronic effects and hydration. Although crystal structures have only been determined for a subset of the large number of modifications that were synthesized and analyzed in the oligonucleotide context to date, they have yielded guiding principles for the design of new analogs with tailor‐made properties, including pairing specificity, nuclease resistance, and cellular uptake. And, perhaps less obviously, crystallographic studies of CNAs and synthetic pairing systems have shed light on fundamental aspects of DNA and RNA structure and function that would not have been disclosed by investigations solely focused on the natural nucleic acids.     

Two-dimensional strandness-dependent electrophoresis: a method to characterize single-stranded DNA, double-stranded DNA, and RNA-DNA hybrids in complex samples

We describe two-dimensional strandness-dependent electrophoresis (2D-SDE) for quantification and length distribution analysis of single-stranded (ss) DNA fragments, double-stranded (ds) DNA fragments, RNA-DNA hybrids, and nicked DNA fragments in complex samples. In the first dimension nucleic acid molecules are separated based on strandness and length in the presence of 7 M urea. After the first-dimension electrophoresis all nucleic acid fragments are heat denatured in the gel. During the second-dimension electrophoresis all nucleic acid fragments are single-stranded and migrate according to length. 2D-SDE takes about 90 min and requires only basic skills and equipment. We show that 2D-SDE has many applications in analyzing complex nucleic acid samples including (1) estimation of renaturation efficiency and kinetics, (2) monitoring cDNA synthesis, (3) detection of nicked DNA fragments, and (4) estimation of quality and in vitro damage of nucleic acid samples. Results from 2D-SDE should be useful to validate techniques such as complex polymerase chain reaction, subtractive hybridization, cDNA synthesis, cDNA normalization, and microarray analysis. 2D-SDE could also be used, e.g., to characterize biological nucleic acid samples. Information obtained with 2D-SDE cannot be readily obtained with other methods. 2D-SDE can be used for preparative isolation of ssDNA fragments, dsDNA fragments, and RNA-DNA hybrids.     

用对流电泳提纯兔出血症病毒

兔出血症病毒（ＲＨＤＶ）在ｐＨ８．６的琼脂糖凝胶上电泳,在负极侧有一血凝峰,免疫电镜观察有大硅ＲＨＤＶ颗粒,证实ＲＨＤＶ病毒粒子带正电荷。利用这一特性可用对流电泳提纯病毒。将抗体与病毒颗粒形成的沉淀带切下,作ＳＤＳ－ＰＡＧＥ,经免疫转印出现６条区带,其中６０ｋＤ的ＶＰ_１为主要结构多肽。用免疫复合物提取核酸,以狄高辛标记制成探针,能与病毒核酸和克隆的ＲＨＤＶｃＤＮＡ２．２ｋｂ片段和４．８ｋｂ片段杂交,探针灵敏度达ｐｇ水平。能用病毒核酸作模板制备探针,证实ＲＨＤＶ的核酸为ＤＮＡ。     

Strategies for optimizing DNA hybridization on surfaces

Specific and predictable hybridization of the polynucleotide sequences to their complementary counterparts plays a fundamental role in the rational design of new nucleic acid nanodevices. Generally, nucleic acid hybridization can be performed using two major strategies, namely hybridization of DNA or RNA targets to surface-tethered oligonucleotide probes (solid-phase hybridization) and hybridization of the target nucleic acids to randomly distributed probes in solution (solution-phase hybridization). Investigations into thermodynamic and kinetic parameters of these two strategies showed that hybridization on surfaces is less favorable than that of the same sequence in solution. Indeed, the efficiency of DNA hybridization on surfaces suffers from three constraints: (1) electrostatic repulsion between DNA strands on the surface, (2) steric hindrance between tethered DNA probes, and (3) nonspecific adsorption of the attached oligonucleotides to the solid surface. During recent years, several strategies have been developed to overcome the problems associated with DNA hybridization on surfaces. Optimizing the probe surface density, application of a linker between the solid surface and the DNA-recognizing sequence, optimizing the pH of DNA hybridization solutions, application of thiol reagents, and incorporation of a polyadenine block into the terminal end of the recognizing sequence are among the most important strategies for enhancing DNA hybridization on surfaces.     

体外核酸快速扩增技术的发展和不断创新

利用聚合酶链式反应(PCR)进行的核酸体外扩增是1983年开始发展起来的一项革命性技术,目前已被广泛运用于现代化的农业和医学以及食品工业等领域,特别是在人类认知基因和基因组的过程中,体外核酸扩增技术做出了卓越的贡献。最初,体外核酸扩增技术主要是利用耐高温的DNA聚合酶(Taq酶),这样就使核酸的体外扩增反应可以在热循环中进行。但因需要使用昂贵的设备和消耗大量的电力,其成本和应用范围都受到一定的限制。之后,恒温体外核酸扩增悄然兴起,这改变了传统扩增技术的局限性,使核酸的体外扩增更加简单和方便。重组酶介导扩增(RAA)法是一种最新型的恒温体外核酸扩增技术,该系统的显著优点在于它在常温下就能实现DNA解链并快速扩增(15~30min完成),反应快速、专一性好、灵敏度高,还可用于定时定量的结果分析。     

简化核酸提取过程在病原体核酸检测中的应用现状和发展趋势

简化核酸提取过程相较于经典的试剂盒法和全自动核酸提取技术,凭借其简便的操作步骤、便携易用的仪器设备和简单的人员培训等优势在病原体核酸检测中得到广泛应用,为实现病原体核酸的快速检测和现场检测(Point-of-care testing,POCT)奠定了良好的基础。本文比较了不同的核酸提取方法,对简化核酸提取过程的方法学进行综述,讨论其在病原体核酸检测领域中的应用现状,并展望其发展趋势。     

Glycotargeting to improve cellular delivery efficiency of nucleic acids

Nucleic acids bearing glycans of various structures have been under vigorous investigation in the past decade. The carbohydrate moieties of such complexes can serve as recognition sites for carbohydrate-binding proteins—lectins—and initiate receptor-mediated endocytosis. Therefore, carbohydrates can enhance cell targeting and internalization of nucleic acids that are associated with them and thus improve the bioavailability of nucleic acids as therapeutic agents. This review summarizes nucleic acid glycosylation in nature and approaches for the preparation of both non-covalently associated and covalently-linked carbohydrate-nucleic acid complexes.     

在AppleⅡ型微机上实现核酸数据处理的工作程序

本文报道了在AppleⅡ型微机上实现核酸数据处理的一系列工作程序。应用这些程序,可进行核酸数据的贮存、对指定的核酸数据结构的改造、限制性内切酶识别位点的检索、核酸序列至蛋白序列的翻译、相关核酸序列及蛋白序列的同源性比较、氨基酸密码使用频率的统计和基因的启动子结构的初步探索等方面的工作。     

2′-BIS-PYRENE MODIFIED OLIGONUCLEOTIDES: SENSITIVE FLUORESCENT PROBES OF NUCLEIC ACIDS STRUCTURE

A new type of fluorescent nucleic acid probes, 2′-bis-pyrene-modified oligonucleotides, is described. Preparation of these conjugates involves attachment of two pyrene moieties to the 2′-phosphate group introduced into any position within a sequence by solid-phase phosphoramidite synthesis. Good hybridization properties of the 2′-bis-pyrene probes, their nuclease resistance and sensitivity of fluorescence to the type of complementary nucleic acid have been demonstrated.