Stem-loop oligonucleotides as tools for labelling double-stranded DNA

We report on a sequence-specific double-stranded DNA labelling strategy in which a stem-loop triplex forming oligonucleotide (TFO) is able to encircle its DNA target. Ligation of this TFO to either a short hairpin oligonucleotide or a long double-stranded DNA fragment leads to the formation of a topological complex. This process requires the hybridization of both extremities of the TFO to each other on a few base pairs. The effects of different factors on the formation of these complexes have been investigated. Efficient complex formation was observed using both GT or TC TFOs. The stem-loop structure enhances the specificity of the complex. The topologically linked TFO remains associated with its target even under conditions that do not favour triple-helix formation. This approach is sufficiently sensitive for detection of a 20-bp target sequence at the subfemtomolar level. This study provides new insights into the mechanics and properties of stem-loop TFOs and their complexes with double-stranded DNA targets. It emphasizes the interest of such molecules in the development of new tools for the specific labelling of short DNA sequences.     

New tools for oligonucleotide fingerprinting

Oligonucleotide fingerprinting is an attractive, high-throughput complement to tag sequencing methods to determine the spectrum and abundance of genes in cDNA libraries. This method currently relies on the sequential hybridizations of short, radioactively labeled DNA oligonucleotides to clone arrays. Here, we describe a new environment that substantially improves this technology. Fluorescently labeled peptide nucleic acid (PNA) oligonucleotides are used as hybridization probes. Hybridization results are recorded with a large-field, high-resolution laser scanner developed for this purpose. Automated image analysis allows easy handling of large numbers of hybridization images. Signal interference effects, which limit the gridding density in the radioactive approach, are strongly reduced. The sensitivity of the fluorescence detection demonstrated permits the convenient use of nylon membranes. Hybridization data quality is improved, and its generation is substantially accelerated, simplified, and less expensive.     

Formation of N-branched oligonucleotides as by-products in solid-phase oligonucleotide synthesis

During the synthesis of oligonucleotides by the standard phosphoramidite method using 2'-deoxycytidine- derivatized solid support, a side reaction was observed that gave rise to the formation of high molecular weight N-branched oligomers having two identical chains linked to the 3'-terminal 2'-deoxycytidine.Postsynthesis treatment with neat triethylamine trihydrofluoride selectively cleaved the phosphoramidate linkage and converted the N-branched oligomers back to the expected oligonucleotides.     

Met signaling mutants as tools for developmental studies

The Met receptor is widely expressed in embryonic and adult epithelial tissues; its ligand (hepatocyte growth factor/scatter factor, HGF/SF) is expressed in the mesenchymal component of various organs. The generation of hgf and met null mice has revealed an essential role for this ligand-receptor pair in the development of the placenta, liver, and limb muscles. However the early lethality of the null mutants has precluded analysis of Met function in late development. To extend the possible observation period, we generated mutant metalleles of different severity. This was done by impairing the ability of the receptor to transduce the HGF/SF signal, via mutation of consensus sequences in the multifunctional docking site present in the C-terminal tail of the receptor. Mice expressing a Met mutant still active as a kinase, but unable to recruit its effectors, died in mid-gestation with the same phenotype as the metknockout, proving the importance of phosphotyrosine-SH2 interactions in vivo. Mice expressing a Met receptor with partial loss of signaling function survived until birth and revealed novel aspects of HGF/SF-Met function during muscle development.     

Cartridge-based high-throughput purification of oligonucleotides for reliable oligonucleotide arrays

A novel, cartridge-based procedure for the efficient and irreversible detritylation of oligonucleotides is reported. This method, combined with a process for the elimination of depurinated fragments produces, in a highly parallel fashion, oligonucleotides with better purity than those traditionally obtained using reversed-phase high-performance liquid chromotography purification. Our combined detritylation and purification methodology compares favorably with commercial cartridge-based purification systems. The benefits of working with pure oligonucleotides, with regard to higher signal and better signal linearity, are shown in array-based hybridization experiments.     

反义寡聚核苷酸：生理学研究中的新工具

反义寡聚核苷酸（antisenseoligonucleotide,AS-ON）通常是指与体内某RNA或DNA序列具有互补顺序,并能通过碱基配对与互补链杂交,从而影响其转录或翻译过程的核酸片段。AS-ONs技术的近来应用为生理学研究开辟了一条新路,对将来了解基因的功能提供了一种新手段。本文综述了AS-ONs的设计策略、作用机理、修饰和导入方式等基本问题,旨在对AS-ONs的应用提供参考。     

Fungal protoplasts as tools for studies on protein secretion

Summary Protoplasts were prepared from mycelium of Aspergillus niger N-402. Sucrose was used to induce the synthesis and secretion of invertase. Protoplasts secreted 2 forms of invertase, different to those secreted by the mycelium. ¹⁴C mannose was shown to be taken up by protoplasts and incorporated into secreted proteins.     

Morpholino antisense oligonucleotides: tools for investigating vertebrate development

Antisense oligonucleotides provide a promising approach to investigating gene function in vivo, but their ability to offer unambiguous insights into phenotypes has been debated. The recent use of morpholino antisense oligonucleotides in zebrafish embryos may prove a major advance, but rigorous controls are essential.     

Viral vectors as tools for studies of central cardiovascular control

During the last few years physiological genomics has been the most rapidly developing area of physiology. Given the current ease of obtaining information about nucleotide sequences found in genomes and the vast amount of readily available clones, one of the most pertinent tasks is to find out about the roles of the individual genes and their families under normal and pathological conditions. Viral gene delivery into the brain is a powerful tool, which can be used to address a wide range of questions posed by physiological genomics including central nervous mechanisms regulating the cardio-vascular system. In this paper, we will give a short overview of current data obtained in this field using viral vectors and then look critically at the technology of viral gene transfer.     

VNTR derived oligonucleotides as PCR primers for population studies in filamentous fungi

One M13 phage derived and three simple sequence repeat primers were assessed for the development of a standard set of PCR primers for population studies with filamentous fungi. Fungal isolates from five plant or insect pathogenic genera with ascomycete and basidiomycete affinities were screened. Three of the four primers were found to be suitable for generating 'genetic fingerprints' for all of the fungal genera tested. Strain groupings obtained from the individual primers were found to be supportive through constrained two-dimensional cluster analysis.     

Bromodeoxyuridine-labeled viral particles as a tool for visualization of the immediate-early events of human cytomegalovirus infection

We describe here a simple method for labeling the genome of human cytomegalovirus, a large double-stranded DNA virus, with bromodeoxyuridine (BrdU). The labeled DNA was incorporated into viral particles, which were then collected in cell supernatant. To demonstrate the versatility and effectiveness of this method, labeled virions were used to study the immediate-early events of virus-host cell interaction via indirect immunofluorescence microscopy. It is our hope that this new methodology will prove useful in the study of binding, entry and viral genome deposition in diverse virus systems.     

Cellular uptake and intracellular fate of antisense oligonucleotides

Antisense oligonucleotides with sequences complementary to a given genetic target can enter cells in sufficient quantities to selectively inhibit gene expression. Thus, they have a potential therapeutic use in preventing undesirable gene expression in diseases such as cancer and AIDS. However, it is remarkable that these molecules, which have high molecular weights and are often charged, gain entry to cells at all. In this article, we review the possible mechanisms by which oligonucleotides enter cells and their subsequent intracellular fates. We also discuss current approaches for improving cellular uptake and delivery of antisense nucleic acids to their intended targets.     

Condensation of oligonucleotides assembled into nicked and gapped duplexes: potential structures for oligonucleotide delivery

The condensation of nucleic acids into well-defined particles is an integral part of several approaches to artificial cellular delivery. Improvements in the efficiency of nucleic acid delivery in vivo are important for the development of DNA- and RNA-based therapeutics. Presently, most efforts to improve the condensation and delivery of nucleic acids have focused on the synthesis of novel condensing agents. However, short oligonucleotides are not as easy to condense into well-defined particles as gene-length DNA polymers and present particular challenges for discrete particle formation. We describe a novel strategy for improving the condensation and packaging of oligonucleotides that is based on the self-organization of half-sliding complementary oligonucleotides into long duplexes (ca. 2 kb). These non-covalent assemblies possess single-stranded nicks or single-stranded gaps at regular intervals along the duplex backbones. The condensation behavior of nicked- and gapped-DNA duplexes was investigated using several cationic condensing agents. Transmission electron microscopy and light-scattering studies reveal that these DNA duplexes condense much more readily than short duplex oligonucleotides (i.e. 21 bp), and more easily than a 3 kb plasmid DNA. The polymeric condensing agents, poly-l-lysine and polyethylenimine, form condensates with nicked- and gapped-DNA that are significantly smaller than condensates formed by the 3 kb plasmid DNA. These results demonstrate the ability for DNA structure and topology to alter nucleic acid condensation and suggest the potential for the use of this form of DNA in the design of vectors for oligonucleotide and gene delivery. The results presented here also provide new insights into the role of DNA flexibility in condensate formation.     

Novel monodisperse PEG-dendrons as new tools for targeted drug delivery: synthesis, characterization and cellular uptake

Dendrimers, dendrons, and hyperbranched polymers are gaining popularity as novel drugs, imaging agents, and drug delivery systems. They present advantages of well-defined molecular weight, multivalent surfaces, and high drug carrying capacity. Moreover, it is emerging that such architectures can display unique endocytic properties. As poly(ethylene glycol) (PEG) is widely used for protein and drug conjugation, the aim of this study was for the first time to synthesize novel, branched PEG-based architectures, to define their cytotoxicity and, via preparation of Oregon green (OG) conjugates define the effect of structure on their cellular uptake. Five PEG-based dendrons were synthesized using monodisperse Fmoc-amino PEG propionic acid (M(w) = 840) as a monomer, and cadaverine, tris(2-aminoethyl)amine or lysine as the branching moieties. These were diamino,bisPEG (M(w) = 1300); triamino,trisPEG (Mw = 1946); tetraamino,tetraPEG (M(w) = 3956); monocarboxy,diamino,bisPEG (M(w) = 1346); and monocarboxy,tetraamino,tetraPEG (M(w) = 3999). These products had NH(2) or both NH(2) and COOH terminal groups and the identity was verified by amino group analysis and ESI-TOF mass spectroscopy. Purity was determined by HPLC. Representative structures were not toxic towards an endothelial-like cell line (ECV304) at concentrations up to 4 mg/mL (over 72 h). At 37 degrees C, all of the OG-labeled PEG dendrons showed progressive uptake by ECV304 cells, but tetraamino,tetraPEG showed the greatest rate of internalization over the first 20 min. Cellular uptake was inhibited at 4 degrees C, and PEG dendron localization to perinuclear vesicles was confirmed by fluorescence microscopy. These well-defined novel architectures have potential for further development as targetable drug delivery systems or tools for construction of structurally defined modified surfaces.     

Aptamers as tools for target validation

Synthetic nucleic acid ligands, called aptamers, bind to protein targets with high specificity and affinity. They are very potent inhibitors of protein function and their application can greatly enhance the process of target validation and drug development. An important benefit of this technology is the recent development of rapidly identifying these sophisticated ligands for almost any target molecule in multi-parallel, automated workstations. The aptamer technology is thus well-suited to addressing the growing demand for high-throughput analysis and functional validation of potential drug targets. Numerous examples have shown the potency of aptamers in inhibiting the function of proteins in cell culture and in vivo models. The technology is complementary to genetic knockout or siRNA approaches as it provides highly valuable information at the proteomic level. In addition, the aptamer technology has recently been extended to developing aptamer drugs and identifying functionally equivalent small molecule leads.     

Glycopeptides as versatile tools for glycobiology

This review describes the recent advances in the field of glycopeptide and small glycoprotein synthesis. The strategies covered include chemical and chemoenzymatic synthesis, native chemical ligation (NCL), and expressed chemical ligation. The importance of glycopeptide synthesis is exemplified by giving the reader an overview of how versatile and important these well-defined glycopeptides are as tools in glycobiology.