摘要:

4′-Thio-β-D-oligoribonucleotides (12 mer and 16 mer) containing a mixed base sequence were synthesized via the phosphoramidite solid support approach. These RNA analogs showed very good nuclease resistance as compared with wild-type RNA. Furthermore, 4′-thio-β-D-oligoribonucleotides were shown to hybridize with a complementary DNA or RNA strand to form a duplex or with a DNA hairpin to form a triple helix. 4′-Thio-RNA binds more tightly to its complementary RNA strand than to its complementary DNA strand. A 4′-thio-RNA:RNA duplex is as stable as a 2′-O-methyl-RNA:RNA duplex. 4′-Thio-RNA, however, forms a 4′thio-RNA:DNA:DNA triplex with a stability similar to the corresponding triplex with al

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.167

年代:1995

数据来源: MAL

摘要:

Oligonucleotides are a class of compounds with potential as therapeutics for a variety of clinical applications. Local delivery of oligonucleotides to the arterial wall is a challenging aspect of the development of these therapeutics for restenosis, and herein we report experiments characterizing the uptake and distribution of phosphorothiate oligonucleotides into vascular smooth muscle cells in primary cultures and in rabbit arteries. Primary cultures of smooth muscle cells incubated with rhodamine-oligonucleotides showed uptake only into cytoplasmic vesicles. No nuclear or cytosolic localization was detected. In normal arteries there was no visible tissue or cellular uptake of oligonucleotides after intralumenal administration. However, in balloon-injured arteries there was significant oligonucleotide uptake into the tissue with apparent cytoplasmic delivery to the medial smooth muscle cells, as evinced by intense staining of their nuclei with labeled oligonucleotides. Measurement of FITC-oligonucleotide in artery extracts showed significantly greater uptake in injured, compared with normal arteries. Light and electron microscopic studies demonstrated a correlation between the degree of damage and the amount of uptake. These results demonstrate that oligonucleotides penetrate easily into the arterial wall of balloon-injured arteries and accumulate in the medial smooth muscle cells—the target cells for antirestenosis therapeutics following balloon angioplast

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.175

年代:1995

数据来源: MAL

摘要:

The use of antisense phosphorothioate oligodeoxynucleotides as tools for modulating gene expression represents a novel strategy for designing drugs to treat a variety of diseases. Several factors, including cellular uptake and internalization of the phosphorothioate oligodeoxynucleotide, are important parameters in determining the effectiveness of antisense agents as therapies. We have used cyclodextrin and its analogs as carriers to increase cellular uptake of phosphorothioate oligodeoxynucleotides. The studies were carried out using35Slabeled and fluorescent-labeled phosphorothioate oligodeoxynucleotide in human T cell leukemia H9 cell line. Cellular uptake of phosphorothioate oligodeoxynucleotide in the presence of cyclodextrin was found to be concentration and time dependent. Using various cyclodextrin analogs, e.g., 2-hydroxypropyl β-cyclodextrin (HPCD), hydroxyethyl β-cyclodextrin (HECD), and a mixture of various hydroxypropyl β-cyclodextrins (Encapsin), we observed increases in phosphorothioate oligodeoxynucleotide uptake, up to twofold to threefold in 48 hours. Confocal microscopy studies confirmed that oligonucleotide was present intracellularly. Cyclodextrin itself was not toxic at the concentration used. Cyclodextrins did not seem to affect the efflux of phosphorothioate oligodeoxynucleotide from cells. Stability of phosphorothioate oligodeoxynucleotide against endogenous cellular nucleases remained unchanged in the presence of cyclodextrins. These studies suggest that cyclodextrin and its analogs might be used successfully as carriers for oligonucleotide and analo

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.185

年代:1995

数据来源: MAL

摘要:

The mechanisms and intracellular pathways by which many oligonucleotide analogs enter cells to exert the desired antisense effects are not fully understood and remain a matter of debate. In this study, we describe the synthesis of 5′-digoxigenin-labeled phosphorothioate oligonucleotides and show their use to examine intracellular oligonucleotide distribution within Epstein-Harr virus-transformed B cells. Comparison of digoxigeninlabeled and fluorescein-labeled oligonucleotide distribution shows the same intracellular fate, suggesting that digoxigenin modification does not interfere with intracellular routing. Double immunofluorescence studied by conventional fluorescence and confocal microscopy with antibodies to the labeling molecule and to lysosomeassociated membrane protein indicate that oligonucleotides mainly accumulate in the lysosomal compartment. Digoxigenin labeling offers an alternative to study oligonucleotide uptake and distribution by immunoelectron microscopy. Two different approaches have been studied: immunogold labeling in heavily fixed and resin-embedded cells and immunogold labeling in lightly fixed and cryoultramicrotomy processed cells. The results confirm the major lysosomal accumulation of digoxigenin-labeled oligonucleotides and demonstrate that the antigenic capacity of digoxigenin is not damaged by any of the procedures used. Therefore, the conjugation of the functionalized digoxigenin molecule at the 5′ end of phosphorothioate oligonucleotides provides a new tool in the study of oligonucleotide uptake and intracellular distribution at both cellular and ultrastructural lev

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.193

年代:1995

数据来源: MAL

摘要:

Ribozymes catalytically cleave substrate RNA molecules in a sequence-specific manner. Engineered ribozymes can be developed and introduced into tissue culture cells to regulate gene expression and to inhibit viral replication. We have previously reported on the construction of cell lines that constitutively express a single antiviral ribozyme embedded in a lengthy RNA transcript. These cells exhibited a marked reduction in their ability to support viral infection. Here we report the construction of RNA molecules that contain one or two antiviral ribozymes, each specific for a different cleavage site on the genome of the target virus, lymphocytic choriomeningitis virus (LCMV), and each contained in a self-cleavage cassette comprisingcis-acting ribozymes designed to release the antiviral molecules from the transcript.In vitrostudies showed that both antiviral ribozymes were released properly from the RNAs following cleavage by the flanking ribozymes and that these released ribozymes functioned as expected in cleaving the target virus RNA. These self-cleaving cassettes have been cloned into a retroviral vector downstream of, but in the same transcript as, the chloramphenicol acetyltransferase (CAT) gene. Thus, we hoped to employ CAT as a surrogate marker of ribozyme transcription. Stably transformed cell lines were established. Cleavage by thecis-acting ribozymes was incomplete, as assessed by Northern blot analysis and by the ability of transformed cells to produce infectious retroviral particles. Nevertheless, the antiviral ribozyme sequences exerted effects in tissue culture. LCMV RNA levels in ribozyme-expressing cells were suppressed, and infectious virus yields were decreased by up to 95% compared with normal cells and with cells expressing inverted ribozymes. The antiviral effects correlated with CAT levels, but there was no significant difference between cell lines expressing a single ribozyme and those expressing two.

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.203

年代:1995

数据来源: MAL

摘要:

To better understand the uptake of oligonucleotides into cells, we have studied the labeling of cell surface proteins by an oligonucleotide conjugated to a radiolabeled photoactivatable crosslinker (Denny-Jaffe reagent). When HL60 cells are treated with the conjugate for 2 hours in a medium containing bovine serum albumin (BSA), almost all of the cell-associated label is found in one protein, which we identify as BSA. Cells grown and treated in a serum-free medium do not show this protein, whereas it is plainly seen in cells that are grown in serum-containing medium but then treated in serum-free medium. Overall association of the oligonucleotide with cells is much higher in serum-free medium than in BSA-containing medium, but the oligonucleotide is mostly not protein-associated in the absence of BSA. We conclude that (1) BSA from the medium serves to block overall association of oligonucleotide with cells, and (2) BSA is the main cell surface protein binding oligonucleotides. We discuss the possible role of albumin in endocytic uptake of oligonucleotides in the cell and in the biodistribution of oligonucleotidesin vivo.

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.213

年代:1995

数据来源: MAL

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.219

年代:1995

数据来源: MAL

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.227

年代:1995

数据来源: MAL

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.233

年代:1995

数据来源: MAL