ISSN:1087-2906    

DOI:10.1089/oli.1.1996.6.1

年代:1996

数据来源: MAL

摘要:

Inhibition of gene expression by phosphorothioate oligomers is complex and involves specific and nonspecific mechanisms. Oligomers that contain a G-quartet elicit distinct effectsin vitroandin vivothat are dependent on the context of the G-quartet's occurrence within a sequence. The enzyme telomerase, a ribonucleoprotein, has a stretch of C residues in the RNA template, which are used to add terminal dG-rich telomeric repeats to the ends of chromosomes. Some but not all phosphorothioates containing a G-quartet, depending on the context of occurrence, inhibited telomerase activityin vitro. Non-G-quartet phosphorothioates did not inhibit this activity. Activities of control enzymes, such as reverse transcriptase ortaqpolymerase, were not affected by the G-quartet oligomers. Neither phosphodiester nor chimeric oligomers of a G-quartet-containing oligomer were as potent in inhibition of telomerase activity as phosphorothioate oligomers. These results may provide a molecular target to study the effects of G-quartet-containing oligomers.

ISSN:1087-2906    

DOI:10.1089/oli.1.1996.6.3

年代:1996

数据来源: MAL

摘要:

Overexpression of erbB-2 proto-oncogene has been found in 20%–30% of human breast carcinomas and in most cases correlates with poor clinical prognosis. Using antisense oligonucleotides targeted to the 5‵ cap region of erbB-2RNA, we were able to inhibit erbB-2 protein expression, proliferation, and anchorage-independent growth of breast cancer cells up to 90%. These effects were sequence specific and restricted to cells expressing elevated level of erbB-2 protein. These results support the feasibility of using antisense erbB-2 oligonucleotides to inhibit the progression of erbB-2-overexpressing breast cancer ce

ISSN:1087-2906    

DOI:10.1089/oli.1.1996.6.9

年代:1996

数据来源: MAL

摘要:

We have analyzed expression of anti-HIV-1 hammerhead ribozymes in the context of retroviral vectors. To determine optimal vector designs for ribozyme expression, we compared three vectors, each of which contained the same pair of anti-HIV-1 hammerhead ribozymes in tandem. Despite the presence of vastly different amounts of vector-derived flanking sequences, the ribozymes produced by each vector had similar cleavage activity when assayedin vitro. The ribozyme vectors were packaged into amphotropic virion and used to transduce human CEM T lymphocytes. Analysis by Northern blot and RNAse protection assays demonstrated that the highest steady-state levels of ribozyme-containing transcripts were produced by a vector in which the ribozymes were expressed under transcriptional control of the vector MoMuLV LTR. Despite these differences in the levels of ribozyme transcripts achieved by the vectors, their ability to confer resistance to HIV-1 replication was similar. Therefore, other factors than the absolute levels of ribozymes play a role in determining the effectiveness of ribozyme vectors to inhibit HIV-1. These may include structural features of the transcripts that affect the antisense effects of the ribozyme constructs, the actual catalytic activity of the ribozymes, their RNA folding, the binding of proteins, and the intracellular localization. Greater understanding of these factors may permit more effective application of ribozymes to inhibit gene expression.

ISSN:1087-2906    

DOI:10.1089/oli.1.1996.6.17

年代:1996

数据来源: MAL

摘要:

IE1 and IE3 mRNAs and their protein products (IE110 and IE175, respectively) were detected in HSV-l-infected U937 cells at 4–15 hours postinfection. In transient expression assays with infectious HIV or an HIV-LTR-directed chloramphenicol acetyltransferase construction (HIV-LTRcat), HSV-1 caused HIV activation (86.7%±6.4% conversion). Electrophoretic mobility shift assays with DNA sequences that encompass the LBP-1 binding site revealed increased levels of DNA-protein complex formation with nuclear extracts from HSV-1 infected as compared with uninfected U937 cells. Novel bands were not seen. HSV-1 mutants respectively deleted in IE110 (dl1403) or IE175 (d120) activated HIV as well as wild-type virus. However, HSV-l-mediated activation was inhibited (26% conversion) by simultaneous treatment with oligonucleoside methylphosphonates (ONMP) that specifically inhibit expression of IE110 (IE1TI) or IE175 (IE3TI). ONMP did not inhibit activation when used individually (83.8% and 67.8% conversion with IETI1 and IE3TI, respectively). Combinations of mutant ONMP that do not inhibit IE110 or IE175 expression did not reduce the levels of HSV-1-mediated activation. These findings suggest that HSV genesIE1andIE3can independently activate HIV in monocytic cells and ONMP that target HSVIEgenes can be used to inhibit HIV activati

ISSN:1087-2906    

DOI:10.1089/oli.1.1996.6.25

年代:1996

数据来源: MAL

摘要:

RNA hybridized to 2′-O-methyloligoribonucleotides and incubated in nuclear extracts from HeLa cells is truncated, resulting in a distinct product terminated at the 5′ end of the antisense oligonucleotide. The activity responsible for this effect is not RNase H but rather a novel exonuclease degrading RNA in the 3′ to 5′ direction. The enzyme requires ATP and Mg2+ions. Except for dATP, no other nucleoside triphosphate or nonhydrolyzable ATP analog supports the exonucleolytic activity. In spite of the nuclear origin and activity requirements similar to those required for pre-mRNA splicing, the exonuclease operates with equal efficiency on intron-containing and intronless RNAs, excluding the possibility that it is associated with the splicing ma

ISSN:1087-2906    

DOI:10.1089/oli.1.1996.6.37

年代:1996

数据来源: MAL

摘要:

Three techniques are evaluated for assessing the purity of synthetic oligodeoxyribonucleotides: reversed phase high-performance liquid chromatography (RP-HPLC), polyacrylamide gel-slab electrophoresis (PAGE), and capillary zone electrophoresis in highly concentrated (18%T) entangled polymer networks (CZE). RP-HPLC does not seem to be able to discriminate and resolve the spectrum of failed sequences expected to accompany an oligonucleotide of a given length. The purity data, as given by the manufacturer, are most often close to 100%. PAGE in 20%T matrices, followed by ethidium bromide staining, gives a good resolution of failure sequences and purity assessments decidedly more realistic. CZE in 18% liquid polyacrylamide is able to resolve to baseline all shorter fragments and to give a precise evaluation of the amount of impurities, based on the intrinsic DNA absorbance at 254 nm. Most of the 18 mer oligonucleotides (and of their phosphorothioate derivatives) analyzed by us, as supplied by three different manufacturers, were found to be contaminated by a spectrum of failed and truncated sequences, ranging in size from 7 mer to 17 mer. The purity data rarely exceeded 80% and most often were of the order of 60%–70%. Conditions for a good routine performance of the CZE technique are describe

ISSN:1087-2906    

DOI:10.1089/oli.1.1996.6.47

年代:1996

数据来源: MAL

摘要:

To find more efficacious therapeutic possibilities for treatment of inflammatory disease, we studied the effects of antisense oligonucleotides on interleukin 1β (IL-1β) production of the human macrophage-like U937 cells. U937 cells were incubated with several kinds of oligonucleotides. Total human IL-1β production was determined using an enzyme-linked immunosorbent assay. An antisense phosphorothioate oligonucleotide (S-oligo), complementary to the sequence, including initiation codon of the IL-1β gene, inhibited IL-1β production in a dose-dependent and sequence-specific manner. The effect of the antisense S-oligo was neutralized by mixing with a sense but not with a scramble S-oligo. Cellular uptake of S-oligo scanned with a laser confocal imaging system was time and temperature dependent, and its intracellular distribution was mainly to the cytosols in U937 cells. Human IL-1β antisense S-oligo inhibited IL-1β production of U937 cells, suggesting a potential to reduce some kinds of inflammatory pro

ISSN:1087-2906    

DOI:10.1089/oli.1.1996.6.55

年代:1996

数据来源: MAL

摘要:

In this study we investigated if specific sequence motifs occur with a higher frequency in antisense oligonucleotides than can be expected on the basis of the mRNA composition to get an impression of the importance of these motifs for antisense effects. Computer analysis of 206 antisense oligonucleotides extracted from the literature and from sequence databases, all targeted against human mRNA, was performed. We compared the sequence composition of these oligonucleotides with the average of 100 equally large and randomly selected sequences from sequence databases and of their target mRNA. We found that the frequency of sequence motifs containing GG, CCC, CC, GAC, and CG is significantly higher and TT and that TCC is significantly lower in antisense oligonucleotide sequences than in the randomly selected mRNA sequences. We conclude that there is a bias in the nucleotide composition of antisense oligonucleotides. Some of these biased sequence motifs have been reported to induce nonantisense effects mediated by protein binding. Further analysis of the biologic function of these motifs is necessary to investigate if they should be avoided or incorporated into future designs of therapeutic effective oligonucleotides.

ISSN:1087-2906    

DOI:10.1089/oli.1.1996.6.63

年代:1996

数据来源: MAL

摘要:

An increase in melting temperature for DNA:DNA duplexes had been observed previously (Zhu et al.Antisense Res. Dev.3:349–356, 1993) when an oligo(δ)ornithine moiety was covalently appended to a short oligodeoxynucleotide. We now report the analysis of duplex formation by electrophoretic gel shift analysis. In the particular example studied, an increase in Tmof 4°C was found to correspond to about a fivefold increase in binding constant. A similar enhancement by the appended cationic peptide was observed when the target strand was RNA. The use of a competitive assay format for avoidance of adsorptive loss at low concentrations (<10-7M) of the oligonucleotide-oligo(δ)ornithine conjugate is prese

ISSN:1087-2906    

DOI:10.1089/oli.1.1996.6.69

年代:1996

数据来源: MAL