The design of antisense oligonucleotides containing locked nucleic acids (LNA) was optimized and compared to intensively studied DNA oligonucleotides, phosphorothioates and 2'-O-methyl gapmers. In contradiction to the literature, a stretch of seven or eight DNA monomers in the center of a chimeric DNA/LNA oligonucleotide is necessary for full activation of RNase H to cleave the target RNA. For 2'-O-methyl gapmers a stretch of six DNA monomers is sufficient to recruit RNase H. Compared to the 18mer DNA the oligonucleotides containing LNA have an increased melting temperature of 1.5-4 degrees C per LNA depending on the positions of the modified residues. 2'-O-methyl nucleotides increase the T(m) by only <1 degree C per modification and the T(m) of the phosphorothioate is reduced. The efficiency of an oligonucleotide in supporting RNase H cleavage correlates with its affinity for the target RNA, i.e. LNA > 2'-O-methyl > DNA > phosphorothioate. Three LNAs at each end of the oligonucleotide are sufficient to stabilize the oligonucleotide in human serum 10-fold compared to an unmodified oligodeoxynucleotide (from t(1/2) = approximately 1.5 h to t(1/2) = approximately 15 h). These chimeric LNA/DNA oligonucleotides are more stable than isosequential phosphorothioates and 2'-O-methyl gapmers, which have half-lives of 10 and 12 h, respectively.
Trinucleotide repeats are microsatellite sequences that are polymorphic in length. Their expansion in specific genes underlies a number of neurodegenerative disorders. Using ultraviolet-visible, circular dichroism, nuclear magnetic resonance (NMR) spectroscopies and electrospray ionization mass spectrometry, the structural preferences of RNA molecules composed of two and four repeats of AGG, CGG and UGG in the presence of K+, Na+ and NH4+ were analysed. (AGG)2A, (AGG)4A, p(UGG)2U and p(UGG)4U strongly prefer folding into G-quadruplexes, whereas CGG-containing sequences can adopt different types of structure depending on the cation and on the number of repeats. In particular, the two-repeat CGG sequence folds into a G-quadruplex in potassium buffer. We also found that each G-quadruplex fold is different: A:(G:G:G:G)A hexads were found for (AGG)2A, whereas mixed G:C:G:C tetrads and U-tetrads were observed in the NMR spectra of G(CGG)2C and p(UGG)2U, respectively. Finally, our NMR study highlights the influence of the strand sequence on the structure formed, and the influence of the intracellular environment on the folding. Importantly, we highlight that although potassium ions are prevalent in cells, the structures observed in the HeLa cell extract are not always the same as those prevailing in biophysical studies in the presence of K+ ions.
Glioblastoma multiforme (GBM) accounts for approximately 12-15% of intracranial neoplasms. The GBM remains refractory to therapy because of tumor heterogeneity, local invasion, and non-uniform vascular permeability to drugs. Patients with GBM have the median survival of approximately 8-10 months, and for those cases where tumor recurs, the average time of tumor progression after therapy is only eight weeks. A combination of different treatment modes as surgery and chemo- or/and radiotherapy extend survival only for a short time, if any. Recently, tenascin-C (TN-C) as a dominant epitope in glioblastoma has been discovered. It is transiently expressed during organogenesis, absent or much reduced in most fully developed organs, but reappears under pathological conditions such as infection, inflammation, or tumorigenesis. It was found that the intensity of TN-C staining correlates with the tumor grade and that the strongest staining indicates poor prognosis.
Small-molecule compound-based therapies have provided new insights into cancer treatment against mitochondrial impairment. N6-furfuryladenosine (kinetin riboside, KR) is a purine derivative and an anticancer agent that selectively affects the molecular pathways crucial for cell growth and apoptosis by interfering with mitochondrial functions and thus might be a potential mitotoxicant. Metabolism of cancer cells is predominantly based on the Crabtree effect that relies on glucose-induced inhibition of cell respiration and thus on oxidative phosphorylation (OXPHOS), which supports the survival of cancer cells in metabolic stress conditions. The simplest way to circumvent this phenomenon is to replace glucose with galactose in the culture environment. Consequently, cells become more sensitive to mitochondrial perturbations caused by mitotoxicants. In the present study, we evaluated several cellular parameters and investigated the effect of KR on mitochondrial functions in HepG2 cells forced to rely mainly on OXPHOS. We showed that KR in the galactose environment is a more potent apoptosis-inducing agent. KR decreases the mitochondrial membrane potential, reduces glutathione level, depletes cellular ATP, and induces reactive oxygen species (ROS) production in the OXPHOS state, leading to the loss of cell viability. Taken together, these results demonstrate that KR directly acts on the mitochondria to limit their function and that the sensitivity of cells is dependent on their ability to cope with energetic stress.
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