摘要:

In an attempt to assay the capability of various oligonucleotides to inhibit gene transcriptionin vivothrough triplex formation, we developed a cellular system employing transfection of a reporter plasmid and putative triplex-forming oligonucleotides targeted to Sp1-binding sites contained within the SV40 early promoter. Using this approach, we demonstrated that the activity of the reporter enzyme, alkaline phosphatase, was highly dependent on the sequence of the oligonucleotides: oligonucleotides utilizing G:GC triplets, but not C:GC triplets, promoted a dose-dependent decrease in reporter enzyme activity. Evidence of physical interaction between Sp1-binding sites within the SV40 promoter and sequence-specific G-rich oligonucleotides has been demonstrated, suggesting triple-helix formation as the most probable explanation for the inhibitory effect on alkaline phosphatase activity observed for these oligonucleotides.Surprisingly, Southern analysis of isolated nuclear DNA indicates that the differences in alkaline phosphatase activity associated with transfection of the different oligonucleotides appear to correlate with internalized plasmid DNA copy number rather than inhibition of transcription. It is intriguing to postulate the existence of a nuclease that is able to recognize and cleave triple-helical DNA structures. This hypothesis implies the existence of a novel mechanism of gene regulation specific for triplex structures and, presumably, independent of transcription.

ISSN:1050-5261    

DOI:10.1089/ard.1994.4.1

年代:1994

数据来源: MAL

摘要:

Several studies have shown that α-oligonucleotides (α-ONs) are more resistant to degradation by nucleases than are β-oligonucleotides (β-ONs), but a few exceptions have been reported. The present work indicates that the resistance of α-ONs to 3′-exonucleases contained in calf or human sera strongly depends on their 3′-terminal sequence. When the 2 last residues were ...A-G, ...C-A, or ...C-T, the degradation rates in tissue culture media with fetal calf serum were similar to those observed for β-ONs, but stabilization factors up to 200 were observed when the 2 last residues were ...T-C, ...A-C, or ...C-C, and intermediate stabilization factors were found with ...G-A, ...T-A, or ...T-T terminal sequences. Other data confirm that α-ONs are significantly more resistant than β-ONs to purified 5′-exo- and endonucleases as well as to 3′-exonucleases contained in snake venom or CEM cell lysates, but suggest that sequence specificity may also exist in these media. These findings, which are consistent with literature data and explain the behavior of the aforementioned exceptions, also emphasize that the stability observed in the presence of purified nucleases cannot be extrapolated to sera. Other results show that handling, heat inactivation, and storage conditions have little effect on the 3′-exonuclease activity of serum-containing media, but can completely modify the nuclease activities of cell extracts. This study, which was carried out by means of the accurate "on-line ISRP cleaning" HPLC technique, brings new insights to the general problem of oligonuc

ISSN:1050-5261    

DOI:10.1089/ard.1994.4.9

年代:1994

数据来源: MAL

摘要:

Moloney murine leukemia virua (MoMuLV)-derived retroviral vectors were engineered to express human immunodeficiency virus type 1 (HIV-1) packaging (ψ) signal and Rev response element (RRE) sequences in either sense or antisense orientation. The RRE sequences were expressed under the control of the herpes simplex virus (HSV) thymidine kinase (tk) promoter fused to the HIV-1trans-activation-responsive (TAR) element, while the ψ signal sequences were expressed under control of the HSVtkpromoter. Both RRE and ψ signal sequences were expressed as part of the 3′ untranslated region of the neomycin phosphotransferase (neo) mRNA. The constructs were used to transfect/infect packaging cell lines and the retroviral vector particles released were used to infect a human CD4+lymphocyte-derived MT4cell line. The stable MT4transformants, harboring proviral vector DNA expressing one to two copies of HIV-1 RRE and ψ signal in either antisense or sense orientation, were each tested for their susceptibility to HIV-1 infection. Compared to the results obtained with the control cells lacking any of the test DNA sequences, the rate of HIV-1 production remained unaltered in RRE1+(sense RNA containing a single copy of RRE) RNA-containing cells, whereas it was delayed in cells expressing both RRE2+(sense RNA containing two copies of RRE) and RRE1-(antisense RNA containing a single copy of RRE) RNA-expressing cells. In cells expressing HIV-1 ψ signal, HIV-1 production remained unaltered in ψ+RNA-expressing cells, whereas it was delayed by up to 30 days in ψ-RNA-expressing cells. These results indicate that retroviral vectors expressing two copies of HIV-1 RRE in sense orientation, a single copy of HIV-1 RRE in antisense orientation, and particularly the HIV-1 ψ signal in antisense orientation can be used to confer HIV-1 r

ISSN:1050-5261    

DOI:10.1089/ard.1994.4.19

年代:1994

数据来源: MAL

摘要:

Temperature dependence of UV and CD spectra of the oligonucleotide 5′-(dT)10-L-(dT)10-L-(dA)10-3′ [tripl(ATT)] [L = -pO(CH2CH20)3p-] in phosphate buffer, pH 7, at various NaCl concentrations and in the presence or absence of 0.01 M MgCl2has been studied. At low oligonucleotide concentrations (2.2 × 10-5M nucleotide concentration) all structural transitions proceed intramolecularly. Tripl(ATT) exists in three forms: as a three-stranded clip (at low temperatures), a double-stranded hairpin (at intermediate temperatures), and as an open strand (at high temperatures). Thermodynamic parameters of the triplex formation depending on the NaCl concentration were calculated. The CD spectra were assigned to the single-, double-, and three-stranded forms. Ethidium bromide (EtBr) binding to the three-stranded clip was studied. Ethidium bromide molecules were shown to intercalate into the triple helix with the stable complex formation (association constant is 106). One molecule of three-stranded clip binds not more than three EtBr molecules. The proposed synthetic model (oligonucleotide blocks coupled by hydroxyalkyl chains) has been shown to be convenient for studies of the physical and chemical properties of the triplex and other multistranded complexes of

ISSN:1050-5261    

DOI:10.1089/ard.1994.4.27

年代:1994

数据来源: MAL

摘要:

The uptake and intracellular distribution of oligonucleotide phosphorothioates (S-oligonucleotides) and their analogs by various cells lines were studied using internally 35S-labeled oligonucleotides. Intracellular accumulation starts in the first 2 hr and reaches a maximum at about 16 hr. A decrease of intracellular concentration of the S-oligonucleotide was observed after 16 hr, probably due to a significant efflux transport. Cellular uptake was dependent on the extracellular concentration. The intracellular concentration was significantly higher than that in the medium. The uptake and the intracellular distribution were different in the various cell lines studied. Comparative studies of the uptake of the S-oligonucleotide and various 3′ end-modified S-oligonucleotides show that end modified S-oligonucleotides with a hydrophobic group significantly increases the uptake. The introduction of methylphosphonothioate linkages at the 3′ end of the S-oligonucleotide also resulted in an increase in upt

ISSN:1050-5261    

DOI:10.1089/ard.1994.4.35

年代:1994

数据来源: MAL

摘要:

An antisense phosphorothioate oligodeoxynucleotide 27-mer complementary to therevgene mRNA of the human immunodeficiency virus (HIV-1) was administered to rats through intravenous injections and subcutaneous infusions in order to investigate the disposition of this compound. In addition, nonlethal toxic responses of the rat were evaluated. A biphasic plasma clearance with t1/2α of 20–25 min and t1/2β of 27–41 hr was observed. Single doses ranging from 35 to 3257 μg were examined, and the plasma concentration and area under the plasma concentration–time curve (AUC) were found to be directly proportional to the dose. Continuous subcutaneous infusion of 50 mg over 28 days was also examined. The oligonucleotide is completely eliminated in the urine over 3 days. Electrophoretic analysis demonstrated that the excreted compound has the same mobility and UV-absorbance profile as the administered compound. Measurement of accumulation and distribution into tissues revealed unique tissue-specific rates and extent of oligonucleotide movement into and out of tissues. Results of the chronic infusion study suggest that uptake into tissue is not saturated, even after 28 days of infusion. Analysis of blood plasma from oligonucleotide-treated animals shows a possible transient elevation in levels of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), but not alkaline phosphatase (AP), γ-glutamyltransferase (GT), and bilirubin. The data collectively support the potential utility of phosphorothioate oligonucleotides as therapeutic agen

ISSN:1050-5261    

DOI:10.1089/ard.1994.4.43

年代:1994

数据来源: MAL

摘要:

Antisense oligonucleotides (ASOs) are capable of blocking the expression of targeted genes and are potential antitumor and antiviral therapeutic agents. The specificity of ASO gene inhibition is compromised when homology to other sequences allows the selected ASO to bind to nontargeted mRNAs. To reduce this nonspecific activity, an ASO should target a sequence that is predicted to be unlikely to occur in other mRNAs. The probability of a sequence being unique can be predicted by determining the genomic frequency of short stretches of sequences contained within the target sequence. Two computer programs, OLIGOMER and HEXAGRAPH, were developed for this analysis. OLIGOMER was used to analyze the genomic frequencies of di-, tri-, and hexamers in more than 24 million nucleotides from 8 different genomes in GenBank. A mathematical model was developed that predicts the genomic frequency of longer oligomers on the basis of the observed frequencies of shorter oligomers. The second program, HEXAGRAPH, was used to graphically display the genomic frequency data of a selected target gene. The computational tools developed in this study may help to design more efficient ASOs by decreasing their nonspecific binding activity.

ISSN:1050-5261    

DOI:10.1089/ard.1994.4.53

年代:1994

数据来源: MAL