Unusual conformation of a 3′-thioformacetal linkage in a DNA duplex

Summary The DNA·DNA duplex ·d(GCGCAAAACGCG) (designated duplex III) containing a 3-thioformacetal (3-TFMA) linkage in the center of the sequence was characterized in detail by two- and three-dimensional homonuclear NMR spectroscopy. The NMR results were analyzed and compared with those of two duplexes of the same sequence: One is an unmodified reference sequence and the other contains a formacetal (OCH₂O) linkage at the central T^T step (designated duplex I and duplex II, respectively). In general, the NMR spectra of duplex III closely resemble those of the analogous duplexes I and II, suggesting an overall B-type structure adopted by the 3-TFMA-modified duplex III. Nonetheless, the detection of several distinct spectral features originating from the protons at the modification site is indicative of a local conformation that is clearly different from the corresponding region in duplexes I and II. The 3-thioformacetal linker, in contrast to the formacetal (FMA) linkage, cannot be accommodated in a conformation usually found in natural nucleic acid duplexes. As a consequence, the 3-TFMA-modified T6 sugar adopts an O4-endo form (an intermediate structure between the usual C2-endo and C3-endo forms). This change is accompanied by a change in the (C4–C3–S3–CH₂) dihedral angle and by subsequent adjustments of other torsion angles along the backbone. Notably, this conformational readjustment at the T6–T7 backbone linkage is localized; its collective result has negligible effect on base-base stacking of the T6 and T7 residues. A close examination of the COSY data in all three duplexes reveals a subtle variation in sugar geometry, with more S-type character adopted by the modified duplexes II and III. The results of this study illustrate that, although the difference between FMA and 3-TFMA linkages is merely in the substitution of the T6(O3) in the former by a sulfur atom in the latter, the stereoelectronic difference in a single atom can induce significant local structural distortion in an otherwise well-structured oligonucleotide duplex.Supplementary material available from the authors: One table containing J₁₂, J₁₂ and J₃₄ of duplexes I, II and III.     

Tracking a protein following dissociation from a protein–protein complex using a split SNAP-tag system

Protein–protein interactions (PPIs) are important for various biological processes in living cells. Several methods have been developed for the visualization of PPIs in vivo; however, these methods are unsuitable for visualization of post-PPI events such as dissociation and translocation. In this study, we applied a split SNAP-tag system for the visualization of post-PPI events. This method enabled tracking of the protein following dissociation from the protein–protein complex. Thus, the split SNAP-tag system should prove to be a useful tool for visualization of post-PPI events.     

DNA methylation dynamics of a maternally methylated DMR in the mouse Dlk1–Dio3 domain

The mouse delta-like homolog 1 and type III iodothyronine deiodinase (Dlk1–Dio3) imprinted domain contains three known paternally methylated differentially methylated regions (DMRs): intergenic DMR (IG-DMR), maternally expressed 3-DMR (Gtl2-DMR), and Dlk1-DMR. Here, we report the first maternally methylated DMR, CpG island 2 (CGI-2), is located approximately 800 bp downstream of miR-1188. CGI-2 is highly methylated in sperm and oocytes, de-methylated in pre-implantation embryos, and differentially re-methylated during post-implantation development. CGI-2, similarly to Gtl2-DMR and Dlk1-DMR, acquires differential methylation prior to embryonic day 7.5 (E7.5). Both H3K4me3 and H3K9me3 histone modifications are enriched at CGI-2. Furthermore, CCCTC-binding factor (CTCF) binds to both alleles of CGI-2 in vivo. These results contribute to the investigation of imprinting regulation in this domain.     

Purification and functional analysis of protein kinase G-1α using a bacterial expression system

3',5' Cyclic guanosine monophosphate (cGMP)-dependent protein kinase G-1α (PKG-1α) is an enzyme that is a target of several anti-hypertensive and erectile dysfunction drugs. Binding of cGMP to PKG-1α produces a conformational change that leads to enzyme activation. Activated PKG-1α performs important roles both in blood vessel vasodilation and in maintaining the smooth muscle cell in a differentiated contractile state. Recombinant PKG-1α has been expressed and purified using Sf9-insect cells. However, attempts at purifying full length protein in a soluble and active form in prokaryotes have thus far been unsuccessful. These attempts have been hampered by the lack of proper eukaryotic protein folding machinery in bacteria. In this study, we report the successful expression and purification of PKG-1α using a genetically engineered Escherichia coli strain, Rosetta-gami 2(DE3), transduced with full-length human PKG-1α cDNA containing a C-terminal histidine tag. PKG-1α was purified to homogeneity using sequential nickel affinity chromatography, gel filtration and ion exchange MonoQ columns. Protein identity was confirmed by immunoblot analysis. N-terminal sequencing using Edman degradation demonstrated that the purified protein was full length. Analysis of enzyme kinetics, using a nonlinear regression curve, identified that, at constant cGMP levels (10μM) and varying ATP concentrations, PKG-1α had a maximal velocity (V(max)) of 5.02±0.25pmol/min/μg and a Michaelis-Menten constant (K(m)) of 11.78±2.68μM ATP. Recent studies have suggested that endothelial function can be attenuated by oxidative and/or nitrosative stress but the role of PKG-1α under these conditions is unclear. We found that PKG-1α enzyme activity was attenuated by exposure to the NO donor, spermine NONOate, hydrogen peroxide, and peroxynitrite but not by superoxide, suggesting that the attenuation of PKG-1α activity may be an under-appreciated mechanism underlying the development of endothelial dysfunction in a number of cardiovascular diseases.     

Immunogold localization of TGFβ 1 protein and mRNA in human skin using a colloidal gold/digoxygenin system

Tissue preservation, and immunogold cytochemical and in-situ hybridization labelling intensities vary according to the preparatory protocols used. We wished to determine which preparative protocols produce optimal preservation, protein and mRNA labelling. Nine combinations of fixative and embedding resin were therefore studied using postembedding immunoelectron microscopy and a novel immunogold digoxygenin in situ hybridization (ISH) system, to quantitate the presence of transforming growth factor beta 1 (TGF 1) protein and message in human skin. The best preservation was observed in tissue fixed in 1% glutaraldehyde and embedded in LR White resin or low acid glycolmethacrylate resin (LA-GMA). Preservation was poor in tissue fixed with 1% glutaraldehyde and fair in 4% paraformaldeyde, when embedded in Unicryl. Ethanediol dehydration coupled with LA-GMA embedding resulted in reasonable preservation. Based on quantitative measures of the labelling density for TGF 1 protein and mRNA, immunogold labelling was adequate with 1% glutaraldehyde fixation coupled with LR White or LA-GMA resins, and also with 4% paraformaldehyde and LR White resin, but was best with ethanediol dehydration and LA-GMA embedding. ISH labelling under basal conditions was best in LA-GMA with 1% glutaraldehyde or 4% paraformaldehyde. The ISH label in tissue fixed with 1% glutaraldehyde and embedded in LA-GMA was significantly increased by treatment with proteinase K. Overall, ethanediol dehydration was associated with a good immunoelectron microscopic (IEM) label while LA-GMA with 1% glutaraldehyde or 4% paraformaldehyde resulted in a consistently detectable ISH label. LA-GMA embedding with 1% glutaraldehyde fixation gave a good result with both IEM and ISH labelling.     

Detection of protein–ligand interactions by NMR using reductive methylation of lysine residues

We show that reductive methylation of proteins can be used for highly sensitive NMR identification of conformational changes induced by metal- and small molecule binding, as well as protein-protein interactions. Reductive methylation of proteins introduces two (13)C-methyl groups on each lysine in the protein of interest. This method works well even when the lysines are not actively involved in the interaction, due to changes in the microenvironments of lysine residues. Most lysine residues are located on the protein exterior, and the exposed (13)C-methyl groups may exhibit rapid localized motions. These motions could be faster than the tumbling rate of the molecule as a whole. Thus, this technique has great potential in the study of large molecular weight systems which are currently beyond the scope of conventional NMR methods.     

In vitro and in vivo assessment of vitamin A encapsulation in a liposome–protein delivery system

Vitamin A (VA) is an essential nutrient needed in small amounts by humans and supports a wide range of biological actions. Retinol, the most common and most biologically active form of VA has also been found to inhibit peroxidation processes in membranes and it has been widely used as an ingredient with pharmaceutical and nutritional applications. VA is a lipophilic molecule, sensitive to air, oxidizing agents, ultraviolet light and low pH levels. For these reasons, it is necessary for VA to be protected against oxidation. Another disadvantage in the application of VA is its low solubility in aqueous media. Both issues (sensitivity and solubility) can be solved by employing encapsulation techniques. Liposomes can efficiently encapsulate lipid-soluble materials, such as VA. The encapsulated materials are protected from environmental and chemical changes. A new liposome/β-lactoglobulin formulation has been developed as a stable delivery system for VA. The aim of this study was the encapsulation of VA into β-lactoglobulin–liposome complexes, recently developed in our laboratory. The in vivo bioavailability characterization of VA was tested after administration in laboratory animals (mice). In this report, we demonstrate that VA could be efficiently entrapped and delivered in a phospholipid–sterol–protein membrane resembling system, a newly synthesized promising carrier. Based on this finding, the phospholipid–sterol–protein membrane resembling system may be one of the promising approaches to enhance VA absorption and to overcome the formulation difficulties associated with lipophilic means. The carrier system described here has huge potential in food fortification applications to treat VA deficiency.     

The effect of glutamate-induced excitotoxicity on DNA methylation in astrocytes in a new in vitro neuron-astrocyte-endothelium co-culture system

Glutamate-induced excitotoxicity is a contributer to many neurological diseases. Astrocytes may represent a new target for treating glutamate-induced excitotoxicity. However, the in vitro culture system that mimics the in vivo microenvironment is lacking. This study aimed to establish a new in vitro co-culture system including neurons, astrocytes, and endothelial cells (NAE), and to investigate the effect of glutamate-induced excitotoxicity on DNA methylation in astrocytes. A NAE co-culture method was created using a Transwell chamber, in which neurons were seeded on the bottom of the lower chamber, endothelial cells were plated on the top membrane, and astrocytes were plated on the bottom membrane of the insert. Glutamate-induced toxicity was induced using glutamate and glycine, and examined using immunofluorescence and lactate dehydrogenase release assay. Global methylation in astrocytes was analyzed, and the expression of DNMT1 and DNMT3a was examined using Western blot analysis. Glutamate treatment induced less neuronal damage in the NAE system compared with the control group in which neurons and astrocytes were cultured alone. Global DNA methylation was increased and the expression of DNMT1 and DNMT3a in astrocytes was increased after glutamate treatment, which was blocked by application of the NMDAR inhibitor MK-801 and the DNMT inhibitor 5-azaC from the endothelial cells. The in vitro ANE culture system is effective for studying glutamate-induced excitotoxicity, and may be used for testing the passage of drugs across the blood–brain barrier. Inhibition of DNA methylation in astrocytes may be a new therapeutic strategy for treating glutamate-induced excitotoxicity.     

Landscape of π–π and sugar–π contacts in DNA–protein interactions

There were 1765 contacts identified between DNA nucleobases or deoxyribose and cyclic (W, H, F, Y) or acyclic (R, E, D) amino acids in 672 X-ray structures of DNA–protein complexes. In this first study to compare π-interactions between the cyclic and acyclic amino acids, visual inspection was used to categorize amino acid interactions as nucleobase π–π (according to biological edge) or deoxyribose sugar–π (according to sugar edge). Overall, 54% of contacts are nucleobase π–π interactions, which involve all amino acids, but are more common for Y, F, and R, and involve all DNA nucleobases with similar frequencies. Among binding arrangements, cyclic amino acids prefer more planar (stacked) π-systems than the acyclic counterparts. Although sugar–π interactions were only previously identified with the cyclic amino acids and were found to be less common (38%) than nucleobase–cyclic amino acid contacts, sugar–π interactions are more common than nucleobase π–π contacts for the acyclic series (61% of contacts). Similar to DNA–protein π–π interactions, sugar–π contacts most frequently involve Y and R, although all amino acids adopt many binding orientations relative to deoxyribose. These DNA–protein π-interactions stabilize biological systems, by up to approximately ?40 kJ mol^?1 for neutral nucleobase or sugar–amino acid interactions, but up to approximately ?95 kJ mol^?1 for positively or negatively charged contacts. The high frequency and strength, despite variation in structure and composition, of these π-interactions point to an important function in biological systems.     

Identification of antisense RNA stem–loops that inhibit RNA–protein interactions using a bacterial reporter system

Many well-characterized examples of antisense RNAs from prokaryotic systems involve hybridization of the looped regions of stem–loop RNAs, presumably due to the high thermodynamic stability of the resulting loop–loop and loop–linear interactions. In this study, the identification of RNA stem–loops that inhibit U1A protein binding to the hpII RNA through RNA–RNA interactions was attempted using a bacterial reporter system based on phage λ N-mediated antitermination. As a result, loop sequences possessing 7–8 base complementarity to the 5′ region of the boxA element important for functional antitermination complex formation, but not the U1 hpII loop, were identified. In vitro and in vivo mutational analysis strongly suggested that the selected loop sequences were binding to the boxA region, and that the structure of the antisense stem–loop was important for optimal inhibitory activity. Next, in an attempt to demonstrate the ability to inhibit the interaction between the U1A protein and the hpII RNA, the rational design of an RNA stem–loop that inhibits U1A-binding to a modified hpII was carried out. Moderate inhibitory activity was observed, showing that it is possible to design and select antisense RNA stem–loops that disrupt various types of RNA–protein interactions.     

Semiquantitative and quantitative analysis of protein–DNA interactions using steady-state measurements in surface plasmon resonance competition experiments

One method commonly used to characterize protein–DNA interactions is surface plasmon resonance (SPR). In a typical SPR experiment, chip-bound DNA is exposed to increasing concentrations of protein; the resulting binding data are used to calculate a dissociation constant for the interaction. However, in cases in which knowledge of the specificity of the interaction is required, a large set of DNA variants has to be tested; this is time consuming and costly, in part because of the requirement for multiple SPR chips. We have developed a new protocol that uses steady-state binding levels in SPR competition experiments to determine protein-binding dissociation constants for a set of DNA variants. This approach is rapid and straightforward and requires the use of only a single SPR chip. Additionally, in contrast to other methods, our approach does not require prior knowledge of parameters such as on or off rates, using an estimate of the wild-type interaction as the sole input. Utilizing relative steady-state responses, our protocol also allows for the rapid, reliable, and simultaneous determination of protein-binding dissociation constants of a large series of DNA mutants in a single experiment in a semiquantitative fashion. We compare our approach to existing methods, highlighting specific advantages as well as limitations.     

High-efficiency recovery of target cells using improved yeast display system for detection of protein–protein interactions

We constructed a high-throughput screening (HTS) system for target cells based on the detection of protein–protein interactions by flow cytometric sorting due to the improvement in the yeast cell surface display system. Interaction model proteins, which are the ZZ domain derived from Staphylococcus aureus and the Fc part of human immunoglobulin G (IgG), were displayed on the yeast cell surface. We achieved a rapid and enhanced expression of these proteins as a result of adopting an appropriate yeast strain and a suitable promoter. The displayed ZZ domain had an ability to bind to rabbit IgG and the displayed Fc part to protein A. These were confirmed by flow cytometry and fluorescence microscopy. Furthermore, the cells displaying the ZZ domain or Fc part were isolated from the model libraries constructed by mixing the control yeast cells with the target yeast cells. The ratio of the target cells was increased from 0.0001% to more than 70% by two cycles of cell sorting. These results indicate that we can achieve a rapid and highly efficient isolation method for the target cells with FACSCalibur and that this method will further extend the application of flow cytometric sorting to library selections.     

Flow injection chemiluminescence determination of acetochlor and cartap-HCl in freshwater samples using an acidic KMnO4–rhodamine-B reaction system

A flow injection (FI) methodology using the acidic potassium permanganate (KMnO₄)–rhodamine-B (Rh-B) reaction with chemiluminescence (CL) detection was established to determine acetochlor and cartap-HCl pesticides in freshwater samples. Experimental parameters were optimized, and Chelex-100 cationic exchanger mini column and solid-phase extraction (SPE) were used as phase separation techniques. Linear calibration curves were observed for the standard solutions of acetochlor and cartap-HCl over the ranges 0.005–2.0 mg L⁻¹ [y = 1155.8x + 57.551, R² = 0.9999 (n = 8)] and 0.005–1.0 mg L⁻¹ [y = 979.76x + 14.491, R² = 0.9998 (n = 8)] with LODs and LOQs of 7.5 × 10⁻⁴ and 8.0 × 10⁻⁴ mg L⁻¹ (3σ blank) and 2.5 × 10⁻³ and 2.7 × 10⁻³ mg L⁻¹ (10σ blank), respectively, with an injection throughput of 140 h⁻¹. These methods were used to estimate acetochlor and cartap-HCl with or without the SPE procedure, respectively, in spiked freshwater samples. Results obtained were not significantly different at a 95% confidence level to those of other reported methods. Recoveries for acetochlor and cartap-HCl were obtained over the ranges 93–112% (RSD = 1.9–3.6%) and 98–109% (RSD = 1.7–3.8%), respectively. The most probable CL reaction mechanism was explored.     

Advances in the study of protein–DNA interaction

Protein-DNA interaction plays an important role in many biological processes. The classical methods and the novel technologies advanced have been developed for the interaction of protein-DNA. Recent developments of these methods and research achievements have been reviewed in this paper.