ISSN:1050-5261    

DOI:10.1089/ard.1995.5.1

年代:1995

数据来源: MAL

摘要:

Oligodeoxynucleotides with (2′-5′) internucleotide linkages have been synthesized on a solid support via standard cyanoethyl phosphoramidite chemistry. This simple change in the oligonucleotide bond connectivity led to unique properties. UV melting temperature experiments indicate that the (2′-′)-oligo-3′-deoxyadenylates, (2′-5′)-3′-dA8and (2′-5′)-3′-dA8(s) phosphorothioate, hybridize selectively to single-stranded RNA but not DNA. The complex (2′-5′)-3′-dA8:poly (U) (Tm= 32°C) was nearly as stable as the natural (3′-5′)-2′-dA8and poly (U) (Tm= 33°C) in 130 mM NaCl, and 10 mM phosphate buffer (pH 7.5). However, no association was observed upon mixing (2′-5′)-3′-dA8and poly (dT). The (2′-5′) linkages also confer greater resistance to exo- and endonucleolytic degradation compared with (3′-5′)-linked oligomers. The rate of degradation of (2′-5′)-3′-dA8was almost four times less than that of (3′-5′)-2′-dA8in cell culture medium containing 10% heat-inactivated fetal calf serum. An increase in stability for (2′-5′)-3′-dA8against endonuclease activity was observed in both cytoplasmic and nuclear extracts. The nucleic acid selectivity of (2′-5′)-oligo-3′-deoxynucleotides may represent an impor

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.3

年代:1995

数据来源: MAL

摘要:

Most mammalian cell types appear to take up antisense oligonucleotides and oligonucleotide analogs from the bathing medium by highly inefficient endocytic mechanisms, and most if not all intracellular oligomer is sequestered in vesicles, still separated by a membrane from the target mRNA. On the other hand, oligonucleotides introduced directly into the cytoplasm by microinjection rapidly accumulate in the cell nucleus. Poor delivery to the designated site of action of antisense oligonucleotides is a major problem limiting their routine use in genetic research and their development as potential therapeutic agents. In view of this difficulty, various means of membrane permeabilization were applied to cultured human leukemia cells in an attempt to enhance intracytoplasmic delivery of fluorescein-tagged oligodeoxynucleotides. The outcome of the manipulations was monitored by flow cytometry and fluorescence microscopy. This work has directly confirmed the conclusion suggested by reported antisense effects, that streptolysin O reversibly permeabilizes the plasma membrane toward oligonucleotides and may be utilized to effect biochemical "microinjection" of these molecules directly into the cytoplasm. KY01 myelogenous leukemia cells treated in this way accumulated over 100-fold higher intracellular levels of oligodeoxynucleotides than in the absence of streptolysin O and, in contrast to the latter case, were observed to concentrate internalized molecules in their nuclei.

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.13

年代:1995

数据来源: MAL

摘要:

The involvement of ribonuclease H (RNase H) in antisense phenomena in intact cells has, to date, only been adequately demonstrated for microinjectedXenopussystems. The significance of RNase H for the antisense effects of oligodeoxynucleotides observed in human and other mammalian cell cultures has remained obscure, in part because of inadequate analytic methods. In this report we show that the "reverse ligation-mediated PCR" (RL-PCR) procedure permits amplification of RNA fragments produced by oligodeoxynucleotide-directed RNase H activity. We have used this procedure to demonstrate RNase H-dependent antisense effects in irreversibly permeabilized (dead) cells and reversibly permeabilized (live) cells.

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.23

年代:1995

数据来源: MAL

摘要:

Direct interaction of oligodeoxynucleotides (ODNs) with proteins represents one of the nonantisense-mediated effects of ODNs. Phosphorothioate-capped ODNs have been shown to inhibit directly thein vitrokinase activity of the chronic myelogenous leukemia-associated protein-tyrosine kinase p210bcr-abl. In this study we have determined the efficacy of this aptameric ODN in a cellular system using the K562 chronic myelogenous leukemia-derived cell line. Significant effects upon cellular phosphotyrosine content, as well as cellular growth in soft agar, are observed. These effects are sequence specific and are not mediated through changes in p21bcr-ablprotein levels. Additional ODNs are described that also reduce cellular phosphotyrosine levels and inhibit growth in soft agar but do not inhibit p210bcr-ablkinase activityin vitro.

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.33

年代:1995

数据来源: MAL

摘要:

A "phosphorothioate triester method" was investigated for the solution-phase synthesis of phosphorothioate oligonucleosides. Using fully protected 3′-phosphorothiolate thymidine bearing O-cyanoethyl and S-2,4-dichlorobenzyl groups as phosphorothioate protecting groups, decathymidine nonaphosphorothioate was efficiently assembled through a blockwise procedure. Two side reactions occurred during the deprotection steps: breakage of internucleoside linkages (1.8% per linkage) and formation of phosphate diester linkages (0.9%). Substitution of the dichlorobenzyl group by the more labile 4-nitrobenzyl S-protecting group reduced the extent of internucleoside bond breakage by one-hal

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.39

年代:1995

数据来源: MAL

摘要:

CC-1065 and U-71,184 bind and hyperstabilize DNA duplexes, but little is known about their effects on nucleic acid duplexes of different structure. A 20 mer DNA sequence (5′-TTACTTCAGTTATGAGACCA) containing a drug binding sequence (5′-AGTTA) was selected as the target sequence, and this was duplexed with complementary antisense sequences containing phosphodiester (PO), phosphorothioate (PS), and methylphosphonate (MP) bonds. The duplexes containing PO or PS bound 2 CC-1065 molecules per duplex, presumably at both the target site and at a lower affinity site (5′-AGTAA) on the antisense strand. The duplex containing MP bound only 1 CC-1065, and all duplexes bound only 1 U-71,184. Both CC-1065 and U-71,184 bound to 20 mer duplexes comprised of oligo(dA)-oligo(dT) (2.5 and 2 drugs per duplex, respectively) and poly(rA)-oligo(dT) (1 drug per 20 base pairs). CC-1065 also bound to duplexes between the PO- or PS-based antisense structures and a complementary synthetic 20 mer RNA sequence, with about 1 drug per duplex in each case. CC-1065 increased theTmfor the 20 mer DNA duplexes 17 to 29°C, and the corresponding values for U-71,184 ranged from 7 to 19°C. CC-1065 raised theTmof oligo(dA)-oligo(dT) and poly(rA)-oligo(dT) 29°C. U-71,184 increased theTmfor oligo(dA)-oligo(dT) 30°C but did not significantly elevate theTmfor the corresponding RNA-DNA duplex. The results show that CC-1065 and U-71,184 are capable of binding and stabilizing a variety of nucleic acid duplexes. These agents or their analogs may become useful ligands for antisense oligonucleotide app

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.49

年代:1995

数据来源: MAL

摘要:

Human megakaryocytic tumor cell lines CHRF-288-11 and HEL (human erythroleukemia) were incubated with antisense phosphodiester (PDE) and phosphorothioate (PS) oligodeoxynucleotides directed against the first six codons of the human serglycin proteoglycan gene. As controls, PDE scrambled and PS sense and scrambled sequences and a probe antisense to a 3′ portion of the coding sequence were used. Treatment with PDE-ODNs did not alter the core protein content of cell or culture medium proteoglycans. Treatment with all the PS-ODNs resulted in loss of the 31 kD) serglycin core protein in the medium, but not the cell-associated proteoglycans, and concomitant appearance of a heavily labeled core protein band at the dye front. This band appears to arise from truncation of the core protein, which leaves the glycosaminoglycan attachment region intact. The higher molecular weight core proteins, which appear to be derived from a betaglycan-like proteoglycan, were not affected by the PDE or PS-ODN treatment. The same effect was seen with or without electroporation, which was used to enhance uptake of the ODNs. Thus treatment of megakaryocytic tumor cells with PS-ODNs appeared to cause a selective degradation of the serglycin core protein in a sequence-independent manner. Degradation most likely occurred intracellularly, because culture supernatants did not degrade exogenously added serglycin proteoglycan, and the presence of superoxide dismutase and catalase in the culture medium during exposure of the cells to the PS-ODNs did not prevent the degradatio

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.59

年代:1995

数据来源: MAL

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.67

年代:1995

数据来源: MAL

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.71

年代:1995

数据来源: MAL