Methods in molecular cardiology: the polymerase chain reaction

Several polymerase chain reaction (PCR) techniques are described in this review to give insight into the potential applications for cardiovascular research. Although PCR can be performed in several ways, all applications are based on the same general principle, the amplification of DNA or RNA by the enzyme polymerase. This amplification provides the opportunity to detect, identify and multiply a single copy of DNA or RNA, in or outside the cell. This powerful technique can be used in several directions of DNA and RNA research resulting in the ability to specifically detect the presence and activity of genes. The use of these techniques in cardiovascular research is discussed here.     

RNAi技术研究进展及其在医学中的应用前景

将双链RNA导入细胞内会引起与其同源基因的沉默,这种现象称为RNA干扰（RNAinterference,RNAi）。就RNAi技术的定义、发现过程、作用机制、产生机理等方面的研究进展作一综述,同时对RNAi技术在医学领域中的应用和存在问题进行了展望。     

RNA interference: the story of gene silencing in plants and humans

RNA interference is an exciting field of functional genomics that can silence viral genes. This property of interfering RNA can be used to combat viral diseases of plants as well as animals and humans. It is a short sequence of nucleic acid that can bind to the mRNA of the gene and interferes the process of its expression. It is diverse in occurrence as well as in applications. It occurs from nematodes to fungi and can cause gene silencing in plants, animals and human beings. Small interfering RNAs are used to silence plant viral genes and in production of therapeutic drugs against Hepatitis or Immuno-deficiency viruses in human. In this review, we will discuss the history, mechanism and applications of RNA interference in plant, animal and human research.     

RNA适体研究及其在医药上的应用

核酸适体（nucleic acid aptamer）是从人工合成的随机单链核酸库中筛选出的特异性与靶物质高度亲和的核酸分子,包括DNA适体和RNA适体. 体外获得核酸适体的方法称为指数富集配体系统进化技术,即SELEX（systematic evolution of ligands by exponential enrichment）. 在SELEX技术获得的核酸适体中,RNA适体因其结构的多样性而具有靶分子广、亲和力高、特异性强等特点. 同时,相比传统抗体,RNA适体分子量小、易改造修饰、制备方便且无免疫原性. 因此,RNA适体在基础研究、临床诊断、药物研制等方面展现了广阔的应用前景. 本文综述了RNA适体的产生、特点、作用方式、优势与局限性,并详细介绍了其在医药研究领域的应用.     

Production in Saccharomycescerevisiae of MS2 virus-like particles packaging functional heterologous mRNAs

Recently, DNA bacteriophages (M13, lambda) have been genetically engineered to transfer genes into mammalian cells. Although efficiencies observed are still relatively low, this opens the possibility of using these viruses as a new class of transfection agents not only for fundamental research purposes but also in gene therapy protocols or in other applications like vaccination. In this respect, it has been shown that a lambda bacteriophage engineered to express the hepatitis B surface antigen in mammalian cells could elicit an immune response against this antigen in mice and rabbits without any specific targeting of the bacteriophage. These impressive results would be even more encouraging if they could be obtained with an RNA bacteriophage, as RNA vaccines are preferred over DNA vaccines for safety reasons. Up to now, RNA bacteriophages have never been engineered for gene delivery. In this paper, we have sought to determine whether such a vector could be obtained by engineering the RNA bacteriophage MS2. We show that MS2 can be produced as virus-like particles (VLPs) in Saccharomyces cerevisiae and is able to package functional heterologous mRNAs, provided that these mRNAs contain the MS2 packaging sequence. For instance, linking the MS2 packaging sequence to the human growth hormone (hGH) mRNA enabled the packaging of this particular mRNA in MS2 VLPs. Functionality in eukaryotic systems of packaged mRNAs was confirmed by showing that mRNAs purified from VLPs can be efficiently translated in vitro and in cell cultures. The high stability of MS2 could, therefore, make MS2 VLPs a very powerful carrier for RNA vaccines.     

Ribozymes: A modern tool in medicine

Since the discovery of ribozymes and self-splicing introns, it has been estimated that this biological property of RNA combined with other recombinant DNA technologies would become a tool to combat viral diseases and control oncogenes. These goals seem like a distinct possibility now. However, there is still a lot to be learned about the mobility of RNA inside the cells and the cellular factors that can impede ribozyme action in order to capitalize fully on the targeted RNA inactivation property of ribozymes. The most effective approach to maximize ribozyme function in a complex intracellular environment is to understand as much as possible about the intracellular fate of the RNA that is being targeted. As new techniques in cell biology become available, such understanding will be less problematic. Fundamental studies of ribozyme structure and mechanism of catalysis are flourishing both at the academic and industrial level and it can be expected that many new developments will continue to take place in these areas in the near future. Here, we review the design, stability and therapeutic application of these technologies illustrating relevant gene targets and applications in molecular medicine. Relevant problems in implementation of the technology, group I and II introns and the differences in applications, ribozyme structure and the application of this technology to virus attack and oncogene downregulation are discussed. Also some of the latest RNA-based technologies such as siRNA, RNA/DNA duplexes and RNA decoys have been introduced.     

A multifunctional bioconjugate module for versatile photoaffinity labeling and click chemistry of RNA

A multifunctional reagent based on a coumarin scaffold was developed for derivatization of naive RNA. The alkylating agent N3BC [7-azido-4-(bromomethyl)coumarin], obtained by Pechmann condensation, is selective for uridine. N3BC and its RNA conjugates are pre-fluorophores which permits controlled modular and stepwise RNA derivatization. The success of RNA alkylation by N3BC can be monitored by photolysis of the azido moiety, which generates a coumarin fluorophore that can be excited with UV light of 320?nm. The azidocoumarin-modified RNA can be flexibly employed in structure-function studies. Versatile applications include direct use in photo-crosslinking studies to cognate proteins, as demonstrated with tRNA and RNA fragments from the MS2 phage and the HIV genome. Alternatively, the azide function can be used for further derivatization by click-chemistry. This allows e.g. the introduction of an additional fluorophore for excitation with visible light.     

Isolation of functional RNA from small amounts of different grape and apple tissues

An efficient, simple, and small-scale procedure for isolating functional ribonucleic acid (RNA) was successfully applied to many different tissues of grape and apple. These woody plants are rich in polyphenolic compounds and polysaccharides that could impair the RNA extraction. The method chosen is based on the use of hot borate buffer at alkaline pH supplemented with several adjuvants and followed by selective precipitations. Starting with only 0.4 g of fresh tissue and working with small tubes (2 mL), we were able to obtain good yields of high-quality RNA suitable for further applications. The procedure can be proposed for many applications, and it is particularly highly recommended when isolating RNA from a large number of samples.     

Multifunctional role of microRNAs in mesenchymal stem cell-derived exosomes in treatment of diseases

Mesenchymal stem cells can be replaced by exosomes for the treatment of inflammatory diseases, injury repair, degenerative diseases, and tumors. Exosomes are small vesicles rich in a variety of nucleic acids [including messenger RNA, Long non-coding RNA, microRNA (miRNA), and circular RNA], proteins, and lipids. Exosomes can be secreted by most cells in the human body and are known to play a key role in the communication of information and material transport between cells. Like exosomes, miRNAs were neglected before their role in various activities of organisms was discovered. Several studies have confirmed that miRNAs play a vital role within exosomes. This review focuses on the specific role of miRNAs in MSC-derived exosomes (MSC-exosomes) and the methods commonly used by researchers to study miRNAs in exosomes. Taken together, miRNAs from MSC-exosomes display immense potential and practical value, both in basic medicine and future clinical applications, in treating several diseases.     

基因工程技术在建立噬菌体抗性机制研究中的应用

在自然发生的噬菌体抗性机制的基础上,应用基因工程技术可以建立广泛的噬菌体抗性机制,为有效解决噬菌体感染问题提供了新的策略。噬菌体编码的抗性、反义RNA技术、自杀陷阱及限制/修饰系统的应用是近年来发展起来的几种抗噬菌体策略,着重对其作用机制、研究进展及其意义作一介绍。同时指出应用基因工程技术构建的噬菌体抗性菌株应用于食品发酵工业中所存在的问题,并对其应用前景作了展望。     

Antisense technology in molecular and cellular bioengineering

Antisense technology is finding increasing application not only in clinical development, but also for cellular engineering. Several types of antisense methods (e.g. antisense oligonucleotides, antisense RNA and small interfering RNA) can be used to inhibit the expression of a target gene. These antisense methods are being used as part of metabolic engineering strategies to downregulate enzymes controlling undesired pathways with regard to product formation. In addition, they are beginning to be utilized to control cell phenotype in tissue engineering constructs. As improved methods for antisense effects that can be externally regulated emerge, these approaches are likely to find increased application in cellular engineering applications.     

Designing and using RNA scaffolds to assemble proteins in vivo

RNA scaffolds are synthetic noncoding RNA molecules with engineered 3D folding harnessed to spatially organize proteins in vivo. Here we provide a protocol to design, express and characterize RNA scaffolds and their cognate proteins within 1 month. The RNA scaffold designs described here are based on either monomeric or multimeric units harboring RNA aptamers as protein docking sites. The scaffolds and proteins are cloned into inducible plasmids and expressed to form functional assemblies. RNA scaffolds find applications in many fields in which in vivo organization of biomolecules is of interest. RNA scaffolds provide extended flexibility compared with DNA or protein scaffolding strategies through programmed modulation of multiple protein stoichiometry and numbers, as well as the proteins' relative distances and spatial orientations. For synthetic biology, RNA scaffolds provide a new platform that can be used to increase yields of sequential metabolic pathways.     

Helical regions in transfer ribonucleic acid

Summary By following the thermal denaturation of transfer RNA (t-RNA) over a wide wavelength range it can be demonstrated that t-RNA dissociates in at least two steps. The lower melting step seems to melt cooperatively, while this can not be stated with confidence about the higher melting region. It is shown that the use of difference spectra of two-stranded transitions gives similar, but different results than the use of difference spectra for three stranded-transitions. It is thus shown that difference spectra can not be used (at least at present) to distinguish strandedness in RNA. Possible applications of the method of difference spectra are discussed.     

Direct adenylation from 5′-OH-terminated oligonucleotides by a fusion enzyme containing Pfu RNA ligase and T4 polynucleotide kinase

5′-Adenylated oligonucleotides (AppOligos) are widely used for single-stranded DNA/RNA ligation in next-generation sequencing (NGS) applications such as microRNA (miRNA) profiling. The ligation between an AppOligo adapter and target molecules (such as miRNA) no longer requires ATP, thereby minimizing potential self-ligations and simplifying library preparation procedures. AppOligos can be produced by chemical synthesis or enzymatic modification. However, adenylation via chemical synthesis is inefficient and expensive, while enzymatic modification requires pre-phosphorylated substrate and additional purification. Here we cloned and characterized the Pfu RNA ligase encoded by the PF0353 gene in the hyperthermophilic archaea Pyrococcus furiosus. We further engineered fusion enzymes containing both Pfu RNA ligase and T4 polynucleotide kinase. One fusion enzyme, 8H-AP, was thermostable and can directly catalyze 5′-OH-terminated DNA substrates to adenylated products. The newly discovered Pfu RNA ligase and the engineered fusion enzyme may be useful tools for applications using AppOligos.     

Opportunities in the design and application of RNA for gene expression control

The past decade of synthetic biology research has witnessed numerous advances in the development of tools and frameworks for the design and characterization of biological systems. Researchers have focused on the use of RNA for gene expression control due to its versatility in sensing molecular ligands and the relative ease by which RNA can be modeled and designed compared to proteins. We review the recent progress in the field with respect to RNA-based genetic devices that are controlled through small molecule and protein interactions. We discuss new approaches for generating and characterizing these devices and their underlying components. We also highlight immediate challenges, future directions and recent applications of synthetic RNA devices in engineered biological systems.     

dsRNA介导植物基因沉默及其应用

植物双链RNA(double stranded RNA,dsRNA)能有效干扰同源基因的表达,近年来已成为功能基因组学研究上的新方法。本文综述了植物dsRNA介导的转基因沉默现象及其特点、分子作用机制、主要介导方法,以及近年来在植物功能基因组学研究上的应用情况。