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J Temsamani

Publications and source records attributed to J Temsamani.

At least 37 records · Page 2Linked to original sources
PubMed1995

Sequence identity of the n-1 product of a synthetic oligonucleotide.

After synthesis and purification of an oligonucleotide, the final product usually contains a low level of n-1 congeneric species. We have sequenced the n-1 population of a 25mer phosphodiester oligonucleotide. The n-1 band was cut from the gel and eluted. Oligonucleotides were tailed with dA and annealed to a dT-tailed plasmid. The recombinant plasmid was ligated and used to transform competent bacteria. Our results show that the n-1 population was heterogeneous. The frequency of truncated nucleotides at the 3'-end was much higher than at the 5'-end of the oligomer. No truncated nucleotides were found in the last four nucleotides at the 5'-end. Our results also show that the chain of oligonucleotides can grow on unreacted sites of a controlled-pore glass support.

Base Sequence↗
PubMed1995

Pharmacokinetics of an anti-human immunodeficiency virus antisense oligodeoxynucleotide phosphorothioate (GEM 91) in HIV-infected subjects.

Human pharmacokinetics of an antisense oligodeoxynucleotide phosphorothioate (GEM 91) developed as an anti-human immunodeficiency virus (HIV) agent was carried out in this study. 35S-Labeled GEM 91 was administered to six HIV-infected individuals by means of 2-hour intravenous infusions at a dose of 0.1 mg/kg. Plasma disappearance curves for GEM 91-derived radioactivity could be described by the sum of two exponentials, with half-life values of 0.18 +/- 0.04 and 26.71 +/- 1.67 hours. The radioactivity in plasma was further evaluated by polyacrylamide gel electrophoresis, showing the presence of both intact GEM 91 and lower molecular weight metabolites. Urinary excretion represented the major pathway of elimination, with 49.15% +/- 6.80% of the administered dose excreted within 24 hours and 70.37% +/- 6.72% over 96 hours after dosing. The radioactivity in urine was associated with lower molecular weight metabolites. No drug-related toxicity was observed.

Adult↗
PubMed1995

Use of cyclodextrin and its derivatives as carriers for oligonucleotide delivery.

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 using 35S-labeled 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 beta-cyclodextrin (HPCD), hydroxyethyl beta-cyclodextrin (HECD), and a mixture of various hydroxypropyl beta-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 analogs.

Amino Acid Sequence↗
PubMed1995

Pharmacokinetics of antisense oligonucleotides.

Antisense oligonucleotides are promising therapeutic agents for the treatment of life-threatening diseases. Intravenous injection of phosphodiester oligonucleotide analogue (P-oligonucleotide) in monkeys shows that the oligonucleotide is degraded rapidly in the plasma with a half-life of about 5 minutes. Administration of a single dose of the phosphorothioate (S-oligonucleotide) in animals by the intravenous route reveals biphasic plasma elimination. An initial short half-life (0.53 to 0.83 hours) represents distribution out of the plasma compartment and a second long half-life (35 to 50 hours) represents elimination from the body. This elimination half-life was similar when the oligonucleotide was administered subcutaneously. In contrast, methylphosphonate oligonucleotides have an elimination half-life of 17 minutes in mice. S-Oligonucleotide was distributed into most of organs of rats and mice. Liver and kidney were the 2 organs with highest uptake of the oligonucleotide. The S-oligonucleotide was primarily excreted in urine. Up to 30% was excreted in the first 24 hours. Repeated daily intravenous injections of a 25-mer S-oligonucleotide into rats showed that the concentrations in the plasma are at steady-state during the 8 days' administration. The data represented here support the potential utility of phosphorothioate and methylphosphonate oligonucleotides as therapeutic agents in vivo.

Animals↗
PubMed1994

Inhibition of in vitro transcription by oligodeoxynucleotides.

Single-stranded short phosphodiester oligodeoxynucleotides have been used to inhibit in vitro T7 transcription system. These oligodeoxynucleotides were complementary to either the 3'-5' or 5'-3' strand of the transcription initiation site of a plasmid containing the gag region of HIV. Our results show that incubation of this plasmid DNA with the oligodeoxynucleotide complementary to the template DNA strand (3'-5', sense oligo) showed efficient inhibition of transcription. Incubation of this plasmid with the oligodeoxynucleotide complementary to the 5'-3' strand (antisense oligo) or a random oligodeoxynucleotide failed to do so. The inhibition of gag transcription was specific since the sense oligo failed to prevent transcription of a plasmid containing U2 RNA sequences. The inhibition of transcription was not limited to T7 RNA polymerase but was also observed with SP6 RNA polymerase.

Bacteriophage T7↗
PubMed1994

Cellular uptake of oligodeoxynucleotide phosphorothioates and their analogs.

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 uptake.

3T3 Cells↗
PubMed1993

A rapid method for quantitation of oligodeoxynucleotide phosphorothioates in biological fluids and tissues.

A sensitive and simple method for the quantitation of oligonucleotide phosphorothioates in biological fluids and tissues is described. This method is based on the extraction of the oligonucleotide from the biological fluids and tissues and immobilization on a nylon membrane. The membrane-bound oligodeoxynucleotide phosphorothioate is then hybridized with labeled complementary oligonucleotide and exposed to X-ray film. The data on the film can be scanned and used to create a standard curve. The sensitivity of detection by the method described here will be useful to monitor the pharmacokinetics of oligonucleotides in bodily fluids and distribution in various tissues. The results indicate that the method is rapid and allows handling of a large number of samples at the same time.

Animals↗
PubMed1993

Self-stabilized antisense oligodeoxynucleotide phosphorothioates: properties and anti-HIV activity.

A new class of oligodeoxyribonucleotides has been designed, referred to here as 'self-stabilized' oligonucleotides. These oligonucleotides have hairpin loop structures at their 3' ends, and show increased resistance to degradation by snake venom phosphodiesterase, DNA polymerase I and fetal bovine serum. The self-stabilized region of the oligonucleotide does not interfere in hybridization with complementary nucleic acids as shown by melting temperature, mobility-shift and RNase H cleavage studies. Various self-stabilized oligonucleotides containing increasingly stable hairpin loop regions were studied for their anti-HIV activity. Pharmacokinetic and stability studies in mice showed increased in vivo persistence of self-stabilized oligonucleotides with respect to their linear counterparts.

Animals↗
PubMed1993

Comparison of in vitro transcriptions using various types of DNA templates.

Improvements in the synthesis of RNA using T7 RNA polymerase and synthetic deoxyoligonucleotides containing the T7 RNA promoter are described. Yields were highest when the double-stranded promoter was generated from a hairpin hybridized to the single-stranded DNA template or when the templates were assembled by ligation of shorter chemically synthesized deoxyoligonucleotides. Our results show that for the synthesis of RNA, templates prepared by ligating smaller deoxyoligonucleotides were as much as 25 times more efficient than those prepared by one-step chemical synthesis.

Base Sequence↗
PubMed1993

Pharmacokinetics, biodistribution, and stability of capped oligodeoxynucleotide phosphorothioates in mice.

Several end-modified oligodeoxynucleotide phosphorothioates (S-oligonucleotides) were studied for their pharmacokinetics, biodistribution, excretion, and metabolic stability in vivo after intravenous administration in mice. The overall tissue distribution and excretion patterns of these S-oligonucleotides were found to be independent of 5' or 3' end modification studied. However, the 3' end modification proved to be of considerable importance with respect to metabolic stability of the oligonucleotide. In the case of uncapped and 5'-capped S-oligonucleotide, only 50% of intact oligonucleotide was recovered out of the total bioavailable concentration in liver and kidney. In contrast, in the case of 3'-capped oligonucleotides almost all bioavailable concentrations of 3'-capped oligonucleotide was found to be intact in kidney and liver at 24 hr after administration. These results demonstrate that superior pharmaceutical potential can be created by 3'-end modification of oligonucleotide phosphorothioates.

Animals↗
PubMed1991

The U2 small nuclear ribonucleoprotein particle associates with nuclear factors in a pre-mRNA independent reaction.

Northern blot analysis of HeLa cell nuclear extract following electrophoresis in nondenaturing gels revealed that a small proportion of U2 small nuclear ribonucleoprotein (snRNP) displays a low mobility, in confirmation of previous reports. This low mobility form of U2 snRNP (termed LMC, for low mobility complex) also formed in vitro when U2 snRNP present in HeLa cytoplasmic S100 was added to a micrococcal nuclease-treated nuclear extract. Of greater experimental value, we found that the LMC also formed when a T7 U2 RNA transcript was assembled into U2 snRNP in a HeLa cytoplasmic S100 system, followed by its incubation in micrococcal nuclease-treated nuclear extract. LMC formation was ATP-dependent and was specific for U2 snRNP since it was not observed with S100-assembled U1 or U4 snRNPs. RNase H cleavage of U2 snRNP in the nuclear extract with an oligonucleotide complementary to nucleotides 28-42 of U2 RNA, as opposed to micrococcal nuclease treatment, rendered the extract competent to form the LMC, indicating that the nuclear factors responsible for LMC formation reside on endogenous U2 snRNP. LMC formation was not competed by excess U2 RNA but was competed by partially purified native U2 snRNP, providing further evidence that the LMC represents an interaction of nuclear factors with already assembled U2 snRNP. LMC formation did not take place on a mutant U2 snRNP lacking the binding site for the two U2-specific proteins, A' and B", nor on mutant U2 snRNPs lacking nucleotides 34-37 or nucleotides 46-49. Further results revealed that nucleotides 35 and 36 of U2 RNA, but not 34 and 37, are required for LMC formation. These experiments demonstrate a nucleotide sequence-specific interaction of U2 snRNP with nuclear factors in the absence of pre-mRNA. Among the U2 RNA nucleotides involved in the formation of this complex are ones previously implicated in base pairing between U2 RNA and the pre-mRNA lariat branch site. These findings are discussed in the context of the possibility that the LMC is on the spliceosome assembly pathway.

Adenosine Triphosphate↗
PubMed1991

Pharmacokinetics, biodistribution, and stability of oligodeoxynucleotide phosphorothioates in mice.

We describe preliminary studies of the pharmacokinetics, biodistribution, and excretion of an oligodeoxy-nucleotide phosphorothioate ([S]oligonucleotide) in mice. After either intravenous or intraperitoneal administration of a single dose (30 mg/kg of body weight), [S]oligonucleotide (35S-labeled at each internucleotide linkage) was found in most of the tissues for up to 48 hr. About 30% of the dose was excreted in urine within 24 hr, irrespective of the mode of administration; the excreted [S]oligonucleotide was found to be extensively degraded. In plasma, stomach, heart, and intestine, the [S]oligonucleotide was degraded by only 15%, whereas in the kidney and liver degradation was about 50% in 48 hr. The surprising observation was made that chain length extension of administered [S]oligonucleotide occurred in kidney, liver, and intestine. These results provide an initial definition of parameters for the pharmaceutical development of antisense oligonucleotides.

Animals↗
PubMed1991

Biotinylated antisense methylphosphonate oligodeoxynucleotides. Inhibition of spliceosome assembly and affinity selection of U1 and U2 small nuclear RNPs.

Methylphosphonate (PC) backbone oligodeoxynucleotides complementary to the 5'-terminal nucleotides of U1 and U2 small nuclear (sn) RNAs do not elicit RNase H action under conditions in which natural (phosphodiester) oligodeoxynucleotides yield extensive RNase H cleavage. We show here that antisense PC oligonucleotides can mask sites in U1 and U2 snRNPs that are required for spliceosome formation. We further report that biotinylated derivatives of antisense PC oligos can be used for affinity selection of U1 and U2 snRNPs.

Base Sequence↗
PubMed1990

Interplay between U2 snRNP and 3' splice factor(s) for branch point selection on human beta-globin pre-mRNA.

We investigated the interaction of U2 snRNP with the branch-3' splice site region of three human beta-globin pre-mRNAs carrying nearly complete (BamHI RNA), 24 nt (Avall RNA) and 14 nt (Accl RNA) of exon 2. All supported splicing, but mRNAs yields were respectively 2 and 10 times lower for Avall and Accl RNAs than for BamHI. Analysis of RNase T1-resistant fragments immunoprecipitated by an anti-(U2)RNP antibody at early times of the splicing reaction showed that the protection encompasses both the branch point region and the end of the intron in BamHI and Avall, but essentially only the branch point in Accl RNAs. Later on, this protection becomes less detectable in BamHI, is reinforced in Avall and remains poorly detectable in Accl RNAs. Similar experiments performed at late times with an anti-Sm antibody recognizing all snRNPs showed that the end of the intron is protected in all but BamHI RNAs. These results support the conclusion that U2 snRNP binds to a fully efficient precursor (BamHI RNA) through another factor(s) recognizing the 3' splice site (U5 snRNP and the so-called U2AF protein are likely candidates). Either the absence of an initial contact between U2 snRNP and the factor(s) recognizing the end of the intron (Accl RNA) or the unability of this ternary complex to undergo a conformational change (Avall RNA) could render these severely truncated precursors poor substrates. These different situations have consequences on the branch point selection itself. BamHI and Avall RNAs use three functional branch points at early times, the usual A residue at -37 and two U residues at -17 and -22. Accl RNA uses only one branch point at -37. Later on, all three branch points are used at the same rate in Avall, while the usual one prevails in BamHI RNAs.

Base Sequence↗
PubMed1989

Purified U5 small nuclear ribonucleoprotein can relieve the inhibition of spliceosome assembly and splicing by snRNP-free nuclear proteins.

As demonstrated by RNase T1 protection assays at 0 degrees C without ATP, U1 and U5 snRNPs purified by isopycnic centrifugation in cesium chloride bind to the 5' and 3' splice sites of human beta-globin pre-mRNA, respectively. We also devised a saturation-complementation assay and have found that this purified U5 snRNP, unlike U1, successfully competes with snRNP-free fractions of nuclear proteins which inhibit spliceosome assembly and splicing. Restoration of activity requires intact U5 snRNA and correlates with the presence of the 100 Kd intron binding protein (IBP) which we have previously characterized (Tazi et al., 1986, Cell 47, 755-766). Our results are compatible with a model in which the recognition of the 3' splice site by IBP-U5 snRNP is one of the earliest events of the spliceosome assembly. It could organize the structure of the 3' splice site region of the human beta-globin like pre-mRNAs. However, on the basis of results showing that beta-globin and major late adenovirus seem to have different requirements with respect to IBP-U5 snRNP, it appears that some pre-mRNAs could have a native structure that necessitates less if at all IBP-U5.

Centrifugation, Density Gradient↗
PubMed1989

B-B' proteins from small nuclear ribonucleoproteins have an endoribonuclease catalytic domain inactive in native particles.

Native small nuclear ribonucleoproteins (snRNPs) purified by several conventional procedures or reconstituted in vitro have no ribonuclease activity. However, when these same snRNPs are centrifuged in cesium chloride gradients at low [Mg2+] and in the presence of sarkosyl, an endoribonuclease is unmasked at the density of core particles (i.e. containing only the set of low molecular weight proteins common to all snRNPs), while an inhibitory component is released in soluble form. The nature of this inhibitor was not further investigated and the molecular events underlying this inhibition/activation process remained only a matter of speculation. On the other hand, evidence was obtained that the nuclease activity is carried by B-B' on the basis of its comigration with B-B' as well as with two of their cleavage products after SDS/polyacrylamide gel electrophoresis of snRNP proteins. One was identified by a B-B'-specific monoclonal antibody. Another one, especially prominent and migrating between D and E core proteins, was identified as the N-terminal half of B-B' by microsequence analysis. Although tightly associated with core snRNPs, the activity is not dependent upon the presence of an snRNA. For the time being, the functional significance of this nuclease remains entirely elusive.

Amino Acid Sequence↗