Watersoluble synthetic nucleic acid analogs--polyethyleneimine derivatives containing nucleic acid bases--conformation and interactionwith nucleic acids

Water soluble polyethyleneimine derivatives containing nucleic acid bases were found to interact with polynucleotides, DNA, RNA. The conformational change by formation of complex was observed by CD spectra and was discussed with the hypochromicity in UV spectra. The rates of interactions between nucleic acid bases in polymers were slow as shown by UV spectra, but the conformational changes of the polynucleotides were fast as shown by CD spectra. In the case of the uracil derivative (PEI-Hse-Ura), high value of CD spectra [theta] 2.80 = -8.0 x 10(-4) for the complex with DNA might be caused by psi type conformation of DNA.     

Allosteric nucleic acid catalysts

Endowing nucleic acid catalysts with allosteric properties provides new prospects for RNA and DNA as controllable therapeutic agents or as sensors of their cognate effector compounds. The ability to engineer RNA catalysts that are allosterically regulated by effector binding has been propelled by the union of modular rational design principles and powerful combinatorial strategies.     

肽核酸(peptide nucleic acid,PNA)阵列

肽核酸(PNA)以N—(2—氨基乙基)甘氨酸替代DNA分子中的磷酸戊糖骨架。它能特异性地识别与DNA、RNA所形成的杂交体。PNA—DNA、PNA—RNA的热稳定性要比相应的DNA—DNA、DNA—RNA高,而且PNA识别单碱基的能力强于DNA和RNA,使之在微阵列,尤其是SNP检测领域有着广泛的应用前景。本文简述了PNA阵列从探针设计、阵列合成、杂交和检测的全过程。     

Bacteriophage-mediated nucleic acid immunisation

Whole bacteriophage lambda particles, containing reporter genes under the control of the cytomegalovirus promoter (P(CMV)), have been used as delivery vehicles for nucleic acid immunisation. Following intramuscular injection of mice with lambda-gt11 containing the gene for hepatitis B surface antigen (HBsAg), anti-HBsAg responses in excess of 150 mIU ml(-1) were detected. When isolated peritoneal macrophages were incubated with whole lambda particles containing the gene for green fluorescent protein (GFP) under the control of P(CMV), GFP antigen was detected on the macrophage surface 8 h later. Results suggested that direct targeting of antigen-presenting cells by bacteriophage 'vaccines' may occur, leading to enhanced immune responses compared to naked DNA delivery. Bacteriophage DNA vaccines offer several advantages: they do not contain antibiotic resistance genes, they offer a large cloning capacity (approximately 15 kb), the DNA is protected from environmental degradation, they offer the potential for oral delivery, and large-scale production is cheap, easy and extremely rapid.     

Fluorinated peptide nucleic acid

The fluorinated olefinic peptide nucleic acid analogue (F-OPA) monomer containing the base thymine was synthesised in 13 steps. PNAs containing this unit were prepared and their pairing properties assessed by means of UV-melting experiments.     

Amyloid-associated nucleic acid hybridisation

Nucleic acids promote amyloid formation in diseases including Alzheimer's and Creutzfeldt-Jakob disease. However, it remains unclear whether the close interactions between amyloid and nucleic acid allow nucleic acid secondary structure to play a role in modulating amyloid structure and function. Here we have used a simplified system of short basic peptides with alternating hydrophobic and hydrophilic amino acid residues to study nucleic acid - amyloid interactions. Employing biophysical techniques including X-ray fibre diffraction, circular dichroism spectroscopy and electron microscopy we show that the polymerized charges of nucleic acids concentrate and enhance the formation of amyloid from short basic peptides, many of which would not otherwise form fibres. In turn, the amyloid component binds nucleic acids and promotes their hybridisation at concentrations below their solution K(d), as shown by time-resolved FRET studies. The self-reinforcing interactions between peptides and nucleic acids lead to the formation of amyloid nucleic acid (ANA) fibres whose properties are distinct from their component polymers. In addition to their importance in disease and potential in engineering, ANA fibres formed from prebiotically-produced peptides and nucleic acids may have played a role in early evolution, constituting the first entities subject to Darwinian evolution.     

Locked nucleic acid: a potent nucleic acid analog in therapeutics and biotechnology

Locked nucleic acid (LNA) is a class of nucleic acid analogs possessing very high affinity and excellent specificity toward complementary DNA and RNA, and LNA oligonucleotides have been applied as antisense molecules both in vitro and in vivo. In this review, we briefly describe the basic physiochemical properties of LNA and some of the difficulties that may be encountered when applying LNA technology. The central part of the review focuses on the use of LNA molecules in regulation of gene expression, including delivery to cells, stability, unspecific effects, toxicity, pharmacokinetics, and design of LNA oligonucleotides. The last part evaluates LNA as a diagnostic tool in genotyping.     

Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone

Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression.     

Rapid, automated nucleic acid probe assays using silicon microstructures for nucleic acid concentration

A system for rapid point-of-use nucleic acid (NA) analysis based on PCR techniques is described. The extraction and concentration of DNA from test samples has been accomplished utilizing silicon fluidic microchips with high surface-area-to-volume ratios. Short (500 bp) and medium size (48,000 bp) DNA have been captured, washed, and eluted using the silicon dioxide surfaces of these chips. Chaotropic (GuHCl) salt solutions were used as binding agents. Wash and elution agents consisted of ethanol-based solutions and water, respectively. DNA quantities approaching 40 ng/cm2 of binding area were captured from input solutions in the 100-1000 ng/mL concentration range. For dilute samples of interest for pathogen detection, PCR and gel electrophoresis were used to demonstrate extraction efficiencies of about 50 percent, and concentration factors of about 10x using bacteriophage lambda DNA as the target. Rapid, multichannel PCR thermal cycling modules with integrated solid-state detection components have also been demonstrated. These results confirm the viability of utilizing these components as elements of a compact, disposable cartridge system for the detection of NA in applications such as clinical diagnostics, biowarfare agent detection, food quality control, and environmental monitoring.     

Ring-closing metathesis reactions in nucleic acid chemistry-cyclic dinucleotides for targeting secondary nucleic acid structures

Cyclic dinucleotides are synthesized using a ring-closing metathesis protocol and incorporated into oligonucleotides. A stabilization of a three-way junction is observed by an oligodeoxynucleotide containing a central 2'-C to 3'-phosphate connection.     

Redox-active daunomycin-spin-labeled nucleic acid complexes

Interaction studies between daunomycin (DM) and enzymatically spin-labeled nucleic acid duplexes reveal two modes of binding by electron spin resonance (ESR) spectroscopy. At a low drug/nucleotide (D/N) ratio, the drug binds in the intercalative mode with only a slight reduction in base mobility. Saturation in the intercalative mode is achieved at a lower D/N ratio for B' DNA than for B DNA. After full intercalation, further addition of DM seems to destabilize the helix and to allow the formation of redox-active DM stacks complexed to the nucleic acid lattice. These stacks will irreversibly oxidize all the nitroxides covalently bound to the 4- or 5-position of the pyrimidine base. Interactions between DM and spin-labeled single-stranded nucleic acids lead directly to the formation of redox-active complexes, while mixing of the drug with spin-labeled nucleic acid building blocks not incorporated in a nucleic acid lattice causes no ESR signal change. Complete disappearance of the ESR signal of spin-labeled nucleic acids extrapolates to a D/N value which is a constant for a particular lattice system and is independent of spin-labeling content.     

Optimization of nucleic acid sequences

Base sequence influences the structure, mechanics, dynamics, and interactions of nucleic acids. However, studying all possible sequences for a given fragment leads to a number of base combinations that increases exponentially with length. We present here a novel methodology based on a multi-copy approach enabling us to determine which base sequence favors a given structural change or interaction via a single energy minimization. This methodology, termed ADAPT, has been implemented starting from the JUMNA molecular mechanics program by adding special nucleotides, "lexides," containing all four bases, whose contribution to the energy of the system is weighted by continuously variable coefficients. We illustrate the application of this approach in the case of double-stranded DNA by determining the optimal sequences satisfying structural (B-Z transition), mechanical (intrinsic curvature), and interaction (ligand-binding) properties.     

Isotachophoresis of nucleic acid constituents

Determination methods for purine and pyrimidine bases, nucleosides, nucleotides and related compounds using analytical capillary isotachophoresis are reviewed. First, the isotachophoretic characterization of these compounds, as well as methods for sample preparation prior to analysis, and the different ways of detecting unknown substances in complex biological systems are described. Then applications of isotachophoretic analysis in medical diagnosis and biomedical research are reviewed. In particular, the analysis of purines, pyrimidines, nucleosides and related compounds in blood and serum for the diagnosis and treatment of inherited diseases and cancer is described. Selected applications of nucleotide analysis in biomedical research using different tissue extracts are also reviewed, and some examples of nucleotide-dependent enzymic reactions, which were performed by means of analytical isotachophoresis, are presented.