Ultrafast dynamics in polycyclic aromatic hydrocarbons: the key case of conical intersections at higher excited states and their role in the photophysics of phenanthrene monomer.

In this study we reveal the detailed photocycle of a phenanthrene monomer. Phenanthrene serves as a popular building block for supramolecular systems and as an archetypal molecule to study the photochemistry of polycyclic aromatic hydrocarbons. By means of femtosecond time-resolved UV-vis transient absorption spectroscopy and molecular modeling, we found that the first bright transition involves the second excited singlet state, which relaxes toward the lowest excited singlet state with a biphasic internal conversion through a conical intersection region: a fast coherent branching followed by an exceptionally slow (∼ps) incoherent internal conversion. We succeeded to pinpoint the complete relaxation pathways and to extract the relevant parameters, e.g., the branching ratio at the conical intersection and internal conversion rates.

Morphological diversity of supramolecular polymers of DNA-containing oligopyrenes - formation of chiroptically active nanosheets.

The effect of DNA on the morphology of supramolecular polymers assembled from chimeric DNA-pyrene oligophosphodiesters in water is described. The number of deoxynucleotides determines if the self-assembly yields 1D or 2D structures. A single guanosine nucleotide attached to a heptapyrenotide induces chiroptical activity in self-assembled nanosheets.

Inhibition of CD40-mediated endothelial cell activation with antisense oligonucleotides.

BACKGROUND: CD40-CD154 interactions play a pivotal role in the amplification of immune responses and, as such, represent an attractive target for immune intervention in a number of disease indications. We have previously shown that binding of human CD154 expressed on the Jurkat D1.1 cell line to porcine CD40 on pig aortic endothelial cells (PAECs) can lead to up-regulation of vascular cell adhesion molecule (VCAM)-1 and MHC class II. This activation can be completely inhibited by the addition of a monoclonal antibody (mAb) to human CD154. In this study, we explore an alternative approach to blocking this pathway with antisense oligonucleotides (ASOs). METHODS: Ten ASOs were generated on the basis of the porcine CD40 cDNA sequence. The ASOs that were found to reduce CD40 expression on PAECs were analyzed for their ability to reduce CD40-mediated PAEC activation. RESULTS: Four ASOs were found to significantly lower surface expression of porcine CD40 on PAECs 48 hr after transfection. Eight of the ASOs were seen to lead to mRNA cleavage products by ribonuclease protection assay. Of the four ASOs tested in the PAEC activation assay, one (ASO-9) showed a dramatic inhibition of PAEC activation (IC50 approximately 1 nM) results comparable to the use of a blocking mAb. Furthermore, we compared the effect of CD40 ASO on tumor necrosis factor alpha receptor signaling, in which we observed no effect, which confirmed ASO specificity. CONCLUSIONS: These results indicate that a CD40-dependent activation pathway can be inhibited with an ASO with high potency and specificity. ASO could be an attractive alternative therapy to the use of mAbs.

Bcl-xl antisense treatment induces apoptosis in breast carcinoma cells.

Upregulated expression of bcl-xL is involved in the initiation and progression of breast cancer by inhibiting tumor cell apoptosis. Here we describe the use of the 2;-O-methoxy-ethoxy antisense oligonucleotide 4259 targeting nucleotides 687-706 of the bcl-xL mRNA, a sequence that does not occur in the pro-apoptotic bcl-xS transcript, to restore apoptosis in estrogen-dependent and independent breast carcinoma cells. The antisense effect of oligonucleotide 4259 was examined on the mRNA and protein level using real-time PCR and Western blot analysis, respectively, and the induction of cell death was investigated in viability and apoptosis assays. Treatment of MCF7 cells with oligonucleotide 4259 at a concentration of 600 nM for 20 hr decreased bcl-xL mRNA and protein levels by more than 80% and 50%, respectively. This resulted in the induction of apoptosis characterized by mitochondrial cytochrome c release, decrease of mitochondrial transmembrane potential, and the appearance of condensed nuclei in approximately 40% of cells. Moreover, oligonucleotide 4259 efficiently downregulated bcl-xL expression and decreased cell growth in the breast carcinoma cell lines T-47D, ZR-75-1, and MDA-MB-231. Our data emphasize the importance of bcl-xL as a survival factor for breast carcinoma cells and suggest that oligonucleotide 4259 deserves further investigations for use in breast cancer therapy.

A novel bispecific antisense oligonucleotide inhibiting both bcl-2 and bcl-xL expression efficiently induces apoptosis in tumor cells.

Bcl-2 and Bcl-xL are inhibitors of apoptosis frequently overexpressed in solid tumors. The bcl-2 and bcl-xL mRNAs share a region of homology comprising nucleotides 605-624 and 687-706, respectively, which differs by only three nucleotides. This sequence does not occur in the proapoptotic splice variant bcl-xS. To test the possibility that oligonucleotides targeting this region have the potential to down-regulate bcl-2 and bcl-xL expression simultaneously, three 2'-O-methoxy-ethoxy-modified phosphorothioate oligonucleotides were designed. These oligonucleotides differed in the number of mismatches to bcl-2 and bcl-xL and in the number of nucleotides to which the modifications were made. The effects of these oligonucleotides on bcl-2 and bcl-xL expression, as well as their abilities to induce apoptosis, were assessed in small cell and non-small cell lung cancer cell lines expressing different basal levels of bcl-2 and bcl-xL. Although all oligonucleotides down-regulated bcl-2 and bcl-xL expression, oligonucleotide 4625, which has no mismatching nucleotides to bcl-2 but three to bcl-xL, two of which were modified by 2'-O-methoxy-ethoxy residues, showed the strongest bispecific activity on the transcript and protein level. In all cell lines this bispecific activity induced apoptotic cell death, as demonstrated by increased uptake of propidium iodide, a 10-100-fold increase in caspase-3-like protease activity, and nuclear condensation and fragmentation. This is the first report of a bcl-2/bcl-xL bispecific antisense oligonucleotide that deserves attention as a therapeutic compound in lung cancer and other malignancies in which bcl-2 and/or bcl-xL are overexpressed.

Induction of apoptosis in lung-cancer cells following bcl-xL anti-sense treatment.

Over-expression of the anti-apoptotic protein bcl-xL is frequently found in lung cancer where it potentially contributes to tumor development, progression and drug resistance. To override the apoptotic block in lung-adenocarcinoma and small-cell-lung-cancer (SCLC) cells caused by over-expression of bcl-xL, an anti-sense oligodeoxynucleotide was designed targeting a sequence unique to the bcl-xL coding region and not shared by the pro-apoptotic splice variant bcl-xS. Moreover, to improve the biophysical properties of the anti-sense compound, 2;-methoxy-ethoxy modifications were made to selected deoxy-ribose residues. The resulting gapmer oligonucleotide 4259 was tested on lung-adenocarcinoma and SCLC cell lines in vitro. Treatment of the adenocarcinoma cell lines A549 and NCI-H125 and the SCLC cell lines SW2 and NCI-H69 with 600 nM 4259 reduced bcl-xL levels by 70 to 90%. In the lung-adenocarcinoma cell lines, apoptosis was induced, as indicated by caspase-3-like protease activation and nuclear condensation and fragmentation. In contrast, in the SCLC cell lines, no induction of apoptosis could be demonstrated. These findings imply that bcl-xL is a more critical survival factor for lung adenocarcinomas than for SCLC, and suggest the use of oligonucleotide 4259 for therapy of this major sub-type of lung cancer.

Small volume flow probe for automated direct-injection NMR analysis: design and performance.

A detailed characterization of an NMR flow probe for use in direct-injection sample analysis is presented. A 600-MHz, indirect detection NMR flow probe with a 120-microl active volume is evaluated in two configurations: first as a stand-alone small volume probe for the analysis of static, nonflowing solutions, and second as a component in an integrated liquids-handling system used for high-throughput NMR analysis. In the stand-alone mode, (1)H lineshape, sensitivity, radiofrequency (RF) homogeneity, and heat transfer characteristics are measured and compared to conventional-format NMR probes of related design. Commonly used descriptive terminology for the hardware, sample regions, and RF coils are reviewed or defined, and test procedures developed for flow probes are described. The flow probe displayed general performance that is competitive with standard probes. Key advantages of the flow probe include high molar sensitivity, ease of use in an automation setup, and superior reproducibility of magnetic field homogeneity which enables the practical implementation of 1D T2-edited analysis of protein-ligand interactions.

The sequence-specific cleavage of RNA by artificial chemical ribonucleases.

Based on work spanning 50 years, several groups have recently achieved the specific cleavage of RNA by attaching RNA-cleaving chemical moieties to antisense oligonucleotides. Such artificial chemical ribonucleases have potential as a possible next generation of antisense compounds and also as probes for structural and functional investigations of RNA. Different chemical moieties, such as polyamines, imidazoles, and metal complexes, have been used as the catalytic part of the artificial nucleases. To be of practical use as therapeutics, however, the conjugates must fulfil a number of strict requirements, such as ease of preparation, chemical stability, selectivity, nontoxicity, and, for metal complexes, inertness to loss of cation from the ligand. In addition, high cleavage efficiency is essential to overcome short lifetimes of cellular mRNA targets, and the reaction should not depend on additional cofactors. Based on these criteria, we believe that metal complexes, in particular macrocyclic lanthanide complexes, have the best chance of success for said purpose.

Creating RNA bulges: cleavage of RNA in RNA/DNA duplexes by metal ion catalysis.

The manipulation of a single-stranded RNA target by forming different RNA/antisense hybrids demonstrates the possibility of cleaving the RNA strand within duplexes. This was achieved using the sequence composition of the antisense oligonucleotide, an approach that results in various bulges [unpaired base(s)] in the RNA target, which is then cleavable at these specific bulge sites under free metal ion or metal complex catalysis. RNA cleavages promoted by metal ions were performed under mild conditions and characterized by separating the RNA fragments carrying end label. The observed products result from intramolecular transesterification causing RNA strand scission. No detectable cleavage of the RNA was observed with either a fully complementary RNA/antisense hybrid or a bulged base in the antisense strand. A molecular modeling study of the RNA backbone suggests that the local conformation of the RNA backbone at a bulge in such hybrid duplexes greatly facilitates the metal-assisted catalytic cleavage. Endonucleolytic RNA cleavage within an RNA/antisense hybrid by metal complexes attached to the antisense oligonucleotide might lead to a new approach in antisense technology with artificial ribonucleases which operate with catalytic turnover.

Towards artificial ribonucleases: the sequence-specific cleavage of RNA in a duplex.

Lanthanide complexes covalently attached to oligonucleotides have been shown to cleave RNA in a sequence-specific manner. Efficient cleavage, however, is at present limited to single-stranded RNA regions, as RNA in a duplex is considerably more resistant to strand scission. To overcome this limitation, we have designed and synthesised artificial nucleases comprising lanthanide complexes covalently linked to oligodeoxyribonucleotides which cleave a partially complementary RNA at a bulged site, in the duplex region. Strand scission occurs at or near the bulge. Cleavage of the RNA target by the metal complex can be addressed via the major or the minor groove. In an example of a competitive situation, where the cleavage moiety has access to both a bulge and a single-strand region, transesterification at the bulge is favoured. Such artificial ribonucleases may find application as antisense agents and as tools in molecular biology. In addition, the results may have importance for the design of artificial ribonucleases which are able to act with catalytic turnover.

Sequence specific antisense oligonucleotide analog interference with spermidine/spermine N1-acetyltransferase gene expression.

Induction of the polyamine acetylating enzyme, spermidine/spermine N1-acetyltransferase (SSAT), is one of several biochemical effects associated with the antiproliferative action of polyamine analogs such as N1, N11 diethylnorspermine (DENSPM). Findings to date indicate that this complex and extremely potent gene response involves increased gene transcription, stabilization of mRNA, enhanced translation and protein stabilization. In this study, SSAT-directed antisense oligonucleotide analogs (AOs) were studied for their ability to prevent enzyme induction by DENSPM. Nine 18-mer fully phosphorothioate modified AOs targeting the start codon, exon 6, stop codon and polyadenylation regions of the human SSAT mRNA were synthesized and evaluated in MALME-3M human melanoma cells prior to and during a 6 hr treatment with 10 microM DENSPM. The most effective AOs were those targeting sequences in the stop codon region. Of these, AO-82 suppressed DENSPM induction of SSAT activity, enzyme protein and mRNA by 70-80%. The quantitative similarity of these effects suggests AO interference with mRNA stabilization, a property apparently mediated by sequences located in the stop codon region. Growth inhibition by DENSPM in the presence of the terminally phophorothioated analogs of AO-82 remained similar to that produced by DENSPM alone. While it is possible that SSAT induction may not be involved in analog-mediated antiproliferative activity, a more likely interpretation is that the approximately 50% suppression of the enzyme response achieved in growth studies is not sufficient to abrogate growth inhibition.

Efficient sequence-specific cleavage of RNA using novel europium complexes conjugated to oligonucleotides.

BACKGROUND: A general method allowing the selective destruction of targeted mRNA molecules in vivo would have broad application in biology and medicine. Metal complexes are among the best synthetic catalysts for the cleavage of RNA, and covalent attachment of suitable metal complexes to oligonucleotides allows the cleavage of complementary single-stranded RNAs in a sequence-specific manner. RESULTS: Using novel europium complexes covalently linked to an oligodeoxyribonucleotide, we have achieved the sequence-specific cleavage of a complementary synthetic RNA. The complexes are completely resistant to chemical degradation under the experimental conditions. The cleavage efficiency of the conjugate strongly depends on the nature of the linker between the oligonucleotide and the complex. Almost complete cleavage of the RNA target has been achieved within 16 h at 37 degrees C. CONCLUSIONS: The results will be important for improving the efficacy of antisense oligonucleotides and will provide a basis for the design of synthetic RNA restriction enzymes. Conjugates of the kind described here may also find application as chemical probes for structural and functional studies of RNA.

Synthesis and hybridization properties of oligonucleotides containing 2'-O-modified ribonucleotides.

A versatile, general way is described for the introduction of different functional groups into oligonucleotides by means of a simple linker at the 2'-position of the sugar. Nucleotide building blocks carrying lipophilic, intercalating or tertiary amino groups can be placed deliberately at any desired position of oligonucleotides by standard automated oligonucleotide synthesis. Thermal denaturation studies with these oligonucleotides reveal the following general trends: i) Modification with lipophilic n-octyl groups has little if any effect on duplex stability; a destabilizing (lipophilic) substituent is better tolerated at or near the ends than in the middle of the oligo. ii) An intercalating substituent (2-aminoanthraquinone) substantially increases duplex stability. iii) N,N-Dimethyl amino residues also increase duplex stability though to a smaller extent than intercalating residues. iv) Modifications at the 5'-end have a more pronounced influence on the TM than the corresponding 3'-modifications. v) Oligonucleotides modified in such a way show little or no loss in sequence specificity.

Effects of an aldose reductase inhibitor on organic osmotic effectors in rat renal medulla.

Renal medullary cells use sorbitol, betaine, and other organic compounds as osmotic effectors (osmolytes) to balance high extracellular NaCl. Excess sorbitol is also implicated in diabetes complications in several organs including kidneys. To study regulation of renal sorbitol, male Wistar rats were given an aldose reductase inhibitor, sorbinil, at 40 mg.kg-1.day-1 in food to block sorbitol formation from glucose. Inner medullas of kidneys were analyzed for osmolytes by high-performance liquid chromatography and atomic absorption. Animals on sorbinil had significantly reduced medullary sorbitol contents in a group on ad libitum water for 10 days (2.7 mmol/kg wet wt compared with 4.8 in controls) and in an antidiuretic group kept 7 days and an additional 3 days without water (3.8 mmol/kg wet wt compared with 7.2 in antidiuretic controls). In both groups, betaine contents were significantly elevated (9.2 mmol/kg wet wt compared with 5.5 in ad libitum water controls: 6.4 mmol/kg wet wt compared with 4.2 in antidiuretic controls). No other osmolytes differed. Total contents of nonurea organic osmolytes maintained a constant ratio to sodium contents; thus increased betaine concomitant with decreased sorbitol may have maintained constant cell volume. In contrast, in animals kept 21 days on sorbinil, there were significant decreases in urea and inositol contents. However, there were no significant differences in sorbitol or betaine compared with controls, suggesting a compensating increase in sorbitol production or in sorbinil removal.

Single-strand DNA triple-helix formation.

Chemical modification studies provide evidence that single-stranded oligodeoxyribonucleotides can form stable intrastrand triple helices. Two oligonucleotides of opposite polarity were synthesized, each composed of a homopurine-homopyrimidine hairpin stem linked to a pyrimidine sequence which is capable of folding back on the hairpin stem and forming specific Hoogsteen hydrogen bonds. Using potassium permanganate as a chemical modification reagent, we have found that two oligodeoxyribonucleotides of sequence composition type 5'-(purine)8(N)4(pyrimidine)8(N)6(pyrimidine)8-3' and 5'-(pyrimidine)8N6(pyrimidine)8N4(purine)8-3' undergo dramatic structural changes consistent with intrastrand DNA triple-helix formation induced by lowering the pH or raising the Mg2+ concentration. The intrastrand DNA triple helix is sensitive to base mismatches.

Water self-diffusion in the calf lens.

Proton pulsed gradient spin echo (PGSE) nuclear magnetic resonance (NMR) spectroscopy was used to explore the free and restricted non-Brownian nature of lens water self-diffusion in calf lens tissue. At all temperatures investigated the water self-diffusion coefficient (Dw) of the cortical homogenate (25% protein) was 1.6-1.7 times greater than that for the nucleus (42% protein), and 0.3-0.5 times the value of Dw for pure water. The nuclear lens homogenate displayed anomalous temperature dependent water diffusion behavior, i.e. a departure from the smooth monotonic decrease in Dw with decreasing temperature, in the temperature range of 3-5 degrees C. By contrast, no such behavior was observed for cortical homogenate. Analysis of water proton echo attenuation data employing a parallel-plate model of restricted diffusion provided values for the parallel-plate barrier separation and self-diffusion coefficient in the limit of free diffusion. Nuclear material showed a smaller spatially average barrier separation and a significantly stronger barrier separation temperature dependence than cortical material.