ISSN:1087-2906    

DOI:10.1089/oli.1.1997.7.1

年代:1997

数据来源: MAL

摘要:

RNA polymerase III promoters direct the ubiquitous, high-level, expression of small, stable RNAs, such as tRNAs, and thus are attractive candidates for achieving stable expression of small therapeutic (e.g., antiviral) molecules, such as ribozymes or antisense RNAs. In this article, we describe the use of a nonviral vector containing a tRNA promoter to express an antilymphocytic choriomeningitis virus (LCMV) ribozyme (tRNA-Rib5). The chimeric tRNA-ribozyme is specifically and efficiently transcribed by pol III in cell-free extracts, and the resulting transcript has appropriate ribozyme activity. In tissue culture studies, high levels of chimeric transcripts were readily detectable and were transported to the cytoplasm, the site of LCMV replication. Despite accumulation of tRNA-Rib5 in the cytoplasm of stably transformed cell clones, antiviral effects were minimal or absent. The implications of these findings and the potential use of this vector system forin vivostudies requiring the delivery of small molecules are discussed.

ISSN:1087-2906    

DOI:10.1089/oli.1.1997.7.3

年代:1997

数据来源: MAL

摘要:

A 25-mer phosphorothioate oligodeoxynucleotide (GEM® 91) complementary to thegaggene mRNA of HTV-1 virus was administered intravenously (IV) at a dose of 10 mg/kg/day for 8 weeks or 25 mg/kg single dose subcutaneously (SC) to adult Rhesus monkeys. No radioactive markers were used. A capillary gel electrophoresis (CGE) method with UV detection was used to determine the concentration of GEM 91 in plasma and the metabolite profile. The metabolite profile was virtually the same following a single dose of either 10 mg/kg IV or 25 mg/kg SC. A different metabolite profile was observed after 4 or 8 weeks of multiple IV doses of 10 mg/kg/day. The extract was subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) for positive identification. Mass spectrometry confirmed the major metabolic pathwayin vivoto be via 3‵-end exonuclease activity. The extract was then subjected to a hybridization-assisted ligation reaction in which only 5‵-end intact metabolites were labeled. Analysis by CGE with laser-induced fluorescence (LIF) detection allowed each of these metabolites to be quantified with a limit of detection of 1 ppb (ng/ml). MALDI-TOFMS identified components digested from both ends of the DNA. This study demonstrates that the combination of quantitative CGE-LIF and MALDI-TOFMS yields a powerful and unique approach to study the metabolism of phosphorothioate oligonucleot

ISSN:1087-2906    

DOI:10.1089/oli.1.1997.7.13

年代:1997

数据来源: MAL

摘要:

Synthesis of stereoregular 3‵,5‵-protected all-Rp and all-Spmethylphosphonate heterooligonucleotides d(MMTrAPMeTPMeTPMeCPMeTAc) (12) complementary to the fragment of HTV-1 splicing acceptor site was achieved via stereocontrolled formation of internucleotide linkage. The coupling was based on the transesterification ofP-stereodefined monomer type of 5‵-O-monomethoxytrityl-2‵-O-deoxynucleoside 3‵-O-[O-(4-nitrophenyl)methylphosphonate]. The nucleophile was at-butylmagnesium chloride activated 5‵-terniinal hydroxyl function of the growing olligonucleotide chain. This and otherP-homochiral oligomers will be used as building blocks for the synthesis of biologically significant, longer stereoregular oligo

ISSN:1087-2906    

DOI:10.1089/oli.1.1997.7.23

年代:1997

数据来源: MAL

摘要:

The effect of blocking expression of a specific gene with antisense phosphodiester oligodeoxynucleotides on the coordinate regulation of myogenesis was studied. Different regions of both fast and slow troponin C (TnC) mRNAs were targeted for binding of the antisense oligomer. The 5‵-cap region of both mRNAs was found to be the most effective target for inhibiting the expression of these genes. Approximately 40%–60% inhibition of expression of a specific isoform of TnC was achieved. However, inhibition of the TnC expression did not appreciably alter the pattern of myogenesis of mouse C2C12cells. The differentiated murine muscle cells were able to cope with this reduced level of the target gene expression by antisense phosphodiester oligomers. We have also used a phosphorothioate oligomer targeted against a common sequence within the coding region of both fast and slow TnC mRNAs. This oligomer was found to be ineffective in blocking TnC gene express

ISSN:1087-2906    

DOI:10.1089/oli.1.1997.7.31

年代:1997

数据来源: MAL

摘要:

Oligonucleotide derivatives conjugated to a chemical construction with two histamine residues imitating the catalytic center of ribonuclease A have been synthesized. In experiments with the conjugates complementary to the 3‵-end and to the variable loop and the T loop of yeast tRNAPhe, it was shown that the compounds can accomplish sequence-specific cleavage of the target RNA in physiologic condition

ISSN:1087-2906    

DOI:10.1089/oli.1.1997.7.39

年代:1997

数据来源: MAL

摘要:

The stability of stereoregular oligo(nucleoside phosphorothioate)s (PS-oligos) in human plasma has been studied. 3‵-Exonuclease present in human plasma appeared to be RPspecific, that is, it cleaves internucleotide phosphorothioate linkages of [RP]-configuration and not those of [SP]-configuration. Therefore, PS-oligos containing all phosphorothioate internucleotide linkages of [RP]-configuration [RP-PS-oligos]) are more effectively degraded by the enzyme than PS-oligos prepared via nonstereocontrolled methods (so-called random mixture of diastereomers [Mix-PS-oligos]), whereas oligo(nucleoside phosphorothioate)s of [SP]-configuration remain intact. The enzyme activity depends on the sequence of nucleobases. The presence of deoxycytidine units (three or more residues) at the 3‵-end of PS-oligo substrate significantly inhibits the enzyme activ

ISSN:1087-2906    

DOI:10.1089/oli.1.1997.7.43

年代:1997

数据来源: MAL

摘要:

A quantitative theoretical analysis of antisense action is presented in which hyperbolic relationships between the logarithm of antisense oligodeoxynucleotide (AODN) ligand concentration, versus cognate messenger RNA (mRNA) and protein concentrations, are derived under conditions of steady state. This analysis incorporates a dual-compartment kinetic model of gene expression. The antisense dose-response functions yield an apparent equilibrium dissociation constant Kd(with the dimensions of concentration), which provides an index of binding affinity and AODN efficacy. Increases in Kdproduce a parallel right shift in the theoretical dose-response curve. The dose ratio derived from the antisense concentrations that produce a 50% reduction in phenotypical response, or mRNA/protein level (i.e., the ED50) for two agents of differing efficacy, is shown to equal the Kdratio for the respective agents. The parameter Kdprovides a potentially useful and experimentally quantifiable constant for comparing AODN analog efficacy. Application of quantitative antisense dose-response relationships (QADRRs) and Kdshould provide a more rigorous foundation for the experimental evaluation of antisense effects.

ISSN:1087-2906    

DOI:10.1089/oli.1.1997.7.49

年代:1997

数据来源: MAL

摘要:

31P NMR is an extremely valuable tool for oligonucleotide research and development. This brief commentary, which is directed to scientists who do not regularly use31P NMR in their work, attempts to outline some of the principles, considerations, and representative applications of31P NMR spectroscopy in oligonucleotide research and development.

ISSN:1087-2906    

DOI:10.1089/oli.1.1997.7.55

年代:1997

数据来源: MAL