A major obstacle in the development of effective oligonucleotide therapeutics is a lack of understanding about their cytosolic and nuclear penetration. To address this problem, we have applied the chloroalkane penetration assay (CAPA) to oligonucleotide therapeutics. CAPA was used to quantitate cytosolic delivery of antisense oligonucleotides (ASOs) and siRNAs and to explore the effects of a wide variety of commonly used chemical modifications and their patterning. We evaluated potential artifacts by exploring the effects of serum, comparing activity data and CAPA data, and assessing the impact of the chloroalkane tag and its linker chemistry. We also used viral transduction to expand CAPA to the nuclear compartment in epithelial and neuronal cell lines. Using this enhanced method, we measured a 48-h time course of nuclear penetration for a panel of chemically diverse modified RNAs. Moving forward, CAPA will be a useful tool for deconvoluting the complex processes of endosomal uptake, escape into the cytosol, and subcellular trafficking of oligonucleotide therapeutics in therapeutically relevant cell types.
We derive constraints on the mass-temperature relation of galaxy clusters from their observed luminosity-temperature relation and X-ray temperature function. Adopting the isothermal gas in hydrostatic equilibrium embedded in the universal density profile of dark matter halos, we compute the X-ray luminosity for clusters as a function of their hosting halo mass. We find that in order to reproduce the two observational statistics, the mass-temperature relation is fairly well constrained as T gas = (1.5 ∼ 2.0) keV(M vir /10 14 h −1 70 M ⊙ ) 0.5∼0.55 , and a simple self-similar evolution model (T gas ∝ M 2/3 vir ) is strongly disfavored. In the cosmological model that we assume (a ΛCDM universe with Ω 0 = 0.3, λ 0 = 0.7 and h 70 = 1), the derived mass-temperature relation suggests that the mass fluctuation amplitude σ 8 is 0.7-0.8.
DNase X is the first mammalian DNase to be isolated that is homologous to DNase I. In this study, we have examined its function using a novel monoclonal antibody and showed it to be expressed on the cell surface as a glycosylphosphatidylinositolanchored membrane protein. High level expression was observed in human muscular tissues and in myotubes obtained in vitro from RD rhabdomyosarcoma cells. We observed that RD myotubes incorporated a foreign gene, lacZ, by endocytosis but that expression of the encoded coding product, -galactosidase, was strongly inhibited. Overexpression of DNase X inhibited endocytosis-mediated gene transfer, whereas knockdown of DNase X with small interfering RNA had the opposite effect. These results reveal that DNase X provides a cell surface barrier to endocytosis-mediated gene transfer.DNase I is a secretory endonuclease that hydrolyzes DNA to 3Ј-hydroxyl oligonucleotides in the presence of divalent metal ions, such as Ca 2ϩ , Mg 2ϩ , and Mn 2ϩ (1-3). Its physiological significance in body fluids has long been unclear; however, a recent study demonstrates that targeted disruption of murine dnase1 increases the generation of anti-DNA antibodies and the development of a systemic lupus erythematosus-like syndrome (4). These findings indicate that DNase I plays an important role in eliminating extracellular DNA that can cause autoimmune diseases in animals.In early investigations, most divalent cation-dependent DNase activities were regarded as being carried out by DNase I. However, more recent studies revealed the existence of several DNase I-like DNases, DNase X/Xib, DNase ␥/DNAS1L3, and DNAS1L2 (5). DNASEX is located at q28 of the human X chromosome and was the first gene to be found that encoded a protein homologous to DNase I (6 -8).DNase X has an extra hydrophobic stretch at its C terminus, which is regarded as its most outstanding structural feature (5). This hydrophobic domain is conserved among mammalian DNase X proteins, suggesting that it has functional importance (9) and that DNase X might play a unique physiological role.In the current study, we have generated a monoclonal antibody (mAb) 3 specific for human DNase X and used it to characterize the molecule. We have presented evidence that DNase X is a glycosylphosphatidylinositol (GPI)-anchored membrane DNase located on the cell surface and on early endocytic vesicles. Furthermore, we have demonstrated that DNase X hydrolyzes endocytosed extracellular DNA, thereby protecting cells from invasion by foreign genes. EXPERIMENTAL PROCEDURESCell Culture-HeLa S3, COS-7, and CHO-K1 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. Human embryonic rhabdomyosarcoma RD cells, obtained from the Health Science Research Resources Bank of Japan, were maintained in growth medium (GM) (Dulbecco's modified Eagle's medium supplemented with 20% fetal calf serum, 100 units/ml penicillin, and mg/ml streptomycin). To induce myogenic differentiatio...
2'-Deoxyribonucleoside-3'-boranophosphates (nucleotide monomers), including four kinds of nucleobases, were synthesized in good yields by the use of new boranophosphorylating reagents. We have explored various kinds of condensing reagents as well as nucleophilic catalysts for the boranophosphorylation reaction with nucleosides. In the synthesis of dinucleoside boranophosphates, undesirable side reactions occurred at the O-4 of thymine and the O-6 of N2-phenylacetylguanine bases. To avoid these side reactions, additional protecting groups, benzoyl (Bz) and diphenylcarbamoyl (Dpc) groups, were introduced to thymine and guanine bases, respectively. As a result, the condensation reactions proceeded smoothly without any side reactions, and the dimers including four kinds of nucleobases were obtained in excellent yields. In the deprotection of the 5'-DMTr group, Et3SiH was found to be effective as a scavenger for the DMTr cation which caused a P-B bond cleavage. After removal of the other protecting groups by the conventional procedure, four kinds of dinucleoside boranophosphates were obtained in good yields.
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