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D Praseuth

Publications and source records attributed to D Praseuth.

9 recordsLinked to original sources
PubMed1992

A triple helix-forming oligonucleotide-intercalator conjugate acts as a transcriptional repressor via inhibition of NF kappa B binding to interleukin-2 receptor alpha-regulatory sequence.

Oligonucleotide-directed triplex formation within upstream regulatory sequences is envisioned as a potential tool for gene inhibition. However, this approach requires that triple helix-forming oligonucleotides are chemically modified, so that the triplex is stable under physiological conditions. Here, we have compared several chemical modifications of an oligonucleotide, targeted to a natural 15-base pair homopyrimidine.homopurine sequence located in the upstream regulatory region of the gene encoding the interleukin-2 receptor alpha chain (p55, IL-2 R alpha). Methylation of the cytosines strongly stabilized the triplex. Further attachment of an intercalating agent (acridine) dramatically increased the stability of the triplex, as assessed by Tm measurements or by band shift assays. Furthermore, the acridine-derivatized oligonucleotide was more efficient in competing away high affinity DNA-binding proteins, as assessed by restriction enzyme inhibition assays. Using a novel footprinting assay, we have further shown that the interaction of the methylcytosine-substituted, acridine-derivatized oligonucleotide with a plasmidic target, harboring the IL-2 R alpha regulatory region, remains highly sequence specific, occurs at physiological pH and is independent of the superhelicity of the plasmid. Acridine derivatization did not impair the exquisite target specificity of triplex formation, since the derivatized oligonucleotide inhibited the binding of nuclear proteins to the overlapping NF kappa B enhancer sequence on an IL-2 R alpha target and not on the related human immunodeficiency virus long terminal repeat target. Finally, the oligonucleotide inhibited the NF kappa B-dependent tax-induced transcriptional activation of the IL-2 R alpha chloramphenicol acetyltransferase construct in live cells, whereas it did not have any effect on a human immunodeficiency virus long terminal repeat chloramphenicol acetyltransferase construct. We conclude that this modified oligonucleotide acts as a transcriptional repressor for the IL-2 R alpha gene via triple helix formation with regulatory sequences.

Acridines↗
PubMed1990

Inhibition of simian virus 40 DNA replication in CV-1 cells by an oligodeoxynucleotide covalently linked to an intercalating agent.

An octathymidylate covalently linked via its 3'-end to an acridine derivative inhibited the cytopathic effect of Simian Virus SV40 on CV-1 cells in culture. Control experiments revealed that this effect was virus-specific and did not arise as a result of oligonucleotide degradation by nucleases. A photoactive probe was covalently attached to the 5'-end of the oligonucleotide-acridine conjugate. Upon UV-irradiation, photocrosslinking was shown to occur at the A. T-rich region within the viral origin of replication. A local triple helix can form at moderate salt concentrations with two octathymidylate-acridine conjugates bound to the octaadenylate sequence. Alternatively the octathymidylate-acridine conjugate can bind to the major groove of duplex DNA forming a local triple helix. Different mechanisms are discussed to explain the inhibition of viral DNA replication.

Acridines↗
PubMed1988

Sequence-targeted photosensitized reactions in nucleic acids by oligo-alpha-deoxynucleotides and oligo-beta-deoxynucleotides covalently linked to proflavin.

Proflavin was covalently linked to the 3'-end or to the 5'-end of an octadeoxythymidylate. This oligonucleotide was synthesized with either the natural beta-anomer of thymidine or its synthetic alpha-anomer. A polymethylene chain was used to link one of the amino groups of proflavin to a terminal thiophosphate group of the oligonucleotide. A 27-mer oligodeoxynucleotide containing an octadeoxyadenylate sequence was used as a target for the proflavin-substituted octadeoxythymidylates. Upon irradiation with visible light, photo-cross-linking reactions induced the formation of branched species that migrated more slowly than the 27-mer on denaturing polyacrylamide gels. Piperidine treatment of the photo-cross-linked species induced strand breaks in the 27-mer. In addition, proflavin induced photosensitized reactions at guanine residues in the 27-mer sequence which were converted to strand breaks following piperidine treatment. Triple-helix formation by the oligothymidylates with their complementary oligodeoxyadenylate sequence at high salt concentration led to photo-cross-linking and cleavage reactions on both sides of the target sequence. These results show that it is possible to target photosensitized reactions to specific sequences on nucleic acids. This opens new possibilities for site-directed mutagenesis and the development of photoactive anti-messenger oligodeoxynucleotides.

Acridines↗
PubMed1988

Immunoregulatory functions of paf-acether. II. Decrease of CD2 and CD3 antigen expression.

Paf-acether (platelet-activating factor) is a phospholipid initially described as a potent platelet-aggregating compound. It is produced by numerous cell types and is now considered as an important mediator of cell-cell interactions. The effect of paf-acether on the expression of CD2 and CD3, two human T cell surface glycoproteins, was investigated by indirect immunofluorescence and flow cytometry. Paf-acether partially down-regulated, in a time- and dose-dependent manner, CD2 and CD3 but not HLA class I antigen expression on peripheral human T cells and Jurkat cells. Lysophosphatidylcholine, a phospholipid closely related to paf-acether, had no detectable modulatory effect on CD2 and CD3 expression. In addition to CD2/CD3 modulation, paf-acether markedly inhibited T cell proliferative response not only to phytohemagglutinin or concanavalin A but also to anti-CD3 or a stimulatory combination of anti-CD2 monoclonal antibodies. These data demonstrate for the first time that lipid mediators such as paf-acether might be involved in the regulation of the expression of cell surface glycoproteins that are essential in the execution of T cell function.

Antibodies, Monoclonal↗
PubMed1988

Sequence-specific binding and photocrosslinking of alpha and beta oligodeoxynucleotides to the major groove of DNA via triple-helix formation.

A photocrosslinking reagent (p-azidophenacyl) was covalently linked to an octathymidylate synthesized with either the natural (beta) anomer of thymidine or the synthetic (alpha) anomer. The oligothymidylate was further substituted by an acridine derivative to stabilize the hybrid formed with a complementary octadeoxyadenylate sequence via intercalation. A single-stranded 27-mer containing a (dA)8 sequence and a 27-mer duplex containing a (dA.dT)8 sequence were used as targets. Upon UV irradiation, photocrosslinking of the octathymidylate to its target sequence was observed, generating bands that migrated more slowly in denaturing gels. In the 27-mer duplex, both strands were photocrosslinked to the octathymidylate. Upon alkaline treatment of the irradiated samples, cleavage of the 27-mers was observed at specific sites. These reactions were analyzed at different salt concentrations. The location of the cleavage sites allowed us to demonstrate the following. (i) Both alpha and beta oligothymidylates can recognize a DNA double helix containing an oligo(dA).oligo(dT) sequence; the oligothymidylate binds to the major groove of DNA in a parallel orientation with respect to the adenine-containing strand of the DNA double helix. (ii) alpha oligothymidylates form helices with a complementary single-stranded oligodeoxyadenylate; the two strands have a parallel orientation independently of whether or not an intercalating agent is attached to the oligothymidylate. (iii) At low salt concentration, beta oligothymidylates form a double helix with an oligodeoxyadenylate in which, as expected, the two strands are antiparallel; at high salt concentration, a triple helix is formed in which the second oligothymidylate is oriented parallel to the adenine-containing strand. These results show that it is possible to recognize an oligopurine.oligopyrimidine sequence in a DNA double helix via local triple-helix formation and to target photochemical reactions to specific sequences in both double-stranded and single-stranded nucleic acids.

Cross-Linking Reagents↗
PubMed1987

Sequence-specific recognition, photocrosslinking and cleavage of the DNA double helix by an oligo-[alpha]-thymidylate covalently linked to an azidoproflavine derivative.

A 3-azidoproflavine derivative was covalently linked to the 5'-end of an octathymidylate synthesized with the [alpha]-anomers of the nucleoside. Two target nucleic acids were used for this substituted oligo-[alpha]-thymidylate: a 27-mer single-stranded DNA fragment containing an octadeoxyadenylate sequence and a 27-mer duplex containing eight contiguous A.T base pairs with all adenines on the same strand. Upon visible light irradiation the octa-[alpha]-thymidylate was photocrosslinked to the single-stranded 27-mer. Chain breaks were induced at the crosslinked sites upon piperidine treatment. From the location of the cleavage sites on the 27-mer sequence it was concluded that a triple helix was formed by the azidoproflavine-substituted oligo-[alpha]-thymidylate with its complementary oligodeoxyadenylate sequence. When the 27-mer duplex was used as a substrate cleavage sites were observed on both strands after piperidine treatment of the irradiated sample. They were located at well defined positions which indicated that the octathymidylate was bound to the (dA)8.(dT)8 sequence in parallel orientation with respect to the (dA)8-containing strand. Specific binding of the [alpha]-octathymidylate involved local triple strand formation with the duplex (dA)8.(dT)8 sequence. This result shows that it is possible to synthesize sequence-specific molecules which specifically bind oligopurine-oligopyrimidine sequences in double-stranded DNA via recognition of the major groove hydrogen bonding sites of the purines.

Acridines↗
PubMed1987

Double helices with parallel strands are formed by nuclease-resistant oligo-[alpha]-deoxynucleotides and oligo-[alpha]-deoxynucleotides covalently linked to an intercalating agent with complementary oligo-[beta]-deoxynucleotides.

Oligo-[alpha]-thymidylates have been synthesized and covalently linked to an intercalating agent (an acridine derivative) and/or to a p-azidophenacyl group. These molecules bind to a complementary oligo-[beta]-deoxynucleotide. A strong stabilization is obtained by covalent attachment of the acridine derivative at the 5' end of the oligo-[alpha]-deoxynucleotide. Upon excitation of the p-azidophenacyl group with ultraviolet light, the oligo-[alpha]-thymidylate is crosslinked to its target sequence. These crosslinks are converted to chain breaks under alkaline conditions. This allows an unambiguous assignment of the orientation of the two oligonucleotide chains. As expected, beta-beta hybrids have an antiparallel orientation, whereas the two chains of alpha-beta hybrids are parallel independently of whether an intercalating agent is covalently linked to the alpha-oligo-nucleotide. Oligo-[alpha]-thymidylates covalently linked to an acridine derivative are highly resistant to endo- and exonucleases. Therefore, they could be used as anti-messengers to block mRNA translation in vivo under conditions where oligo-[beta]-deoxynucleotides are usually hydrolysed.

Exonucleases↗
PubMedDate not supplied

Sequence-targeted photochemical modifications of nucleic acids by complementary oligonucleotides covalently linked to porphyrins.

Porphyrins linked to oligonucleotides produce various types of photodamage on a complementary target DNA. The observed reactions include oxidation of guanine bases and cross-linking reactions of the oligonucleotide to its target sequence. Guanines located close to the porphyrin macrocycle were the most altered as compared to more remote guanines on the target sequence. No specific reaction was observed when the complexes were dissociated at temperatures above the melting temperature of the oligonucleotide-target hybrid. Both cross-linking and oxidation reactions accounted for ca. 60% modification of the target chains in the complex. Our results show that oligonucleotides covalently linked to porphyrins are efficient systems for inducing irreversible sequence-specific photodamage on a target DNA.

Base Sequence↗