Biomedical subjects

N T Thuong

Publications and source records attributed to N T Thuong.

At least 37 records · Page 2Linked to original sources

PubMed1992

Triple-helix formation by an oligonucleotide containing one (dA)12 and two (dT)12 sequences bridged by two hexaethylene glycol chains.

The triple-helix formation by the oligonucleotide (dA)12-x-(dT)12-x-(dT)12, where x is a hexaethylene glycol group, was investigated by thermal denaturation analysis and circular dichroism spectroscopy. Thermal denaturation analysis showed that this single-stranded oligonucleotide is able to fold back on itself twice to give a triple helix at low temperature. Upon an increase in the temperature, two cooperative transitions were observed: formation of a double-stranded structure with a dangling x-(dT)12 extremity, then formation of a single-stranded coil structure. Due to the intramolecular character of the transition, the triplex is much more stable than that formed by the reference mixture (dA)12 + 2(dT)12. In 0.1 M NaCl, the triplex-to-coil transition occurred at about 30 degrees C whereas the duplex-to-coil was at about 60 degrees C. Upon an increase in the salt, the increase of temperature corresponding to the triplex-to-duplex transition was larger than that of the duplex-to-coil transition. MgCl2 showed higher efficiency than NaCl to promote triplex or duplex formation. The thermodynamic parameters delta H and delta S were determined at various ionic strengths for both transitions. Both the enthalpy change and entropy change associated with triplex-to-duplex transition (Hoogsteen base pairing) were smaller than those associated to the duplex-to-coil transition (Watson-Crick base pairing). When the ionic strength increased, the parameters -delta H and -delta S showed a very small decrease for the duplex-to-coil transition whereas a strong increase was observed with the triplex-to-duplex transition.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1992

A novel strategy for site-directed chemical reactions in single stranded DNA--absorption and NMR spectroscopic studies of model compounds.

A new and simple model enabling a chemical species to be brought to a preselected site in single strand DNA is reported. Two oligonucleotides containing a propanediol linkage were hybridized to their complementary sequences with an extra-base opposite the propanediol derivative. Absorption studies results shown that the addition of a bisacridine derivative strongly increased the stabilities of both duplexes when added in a 1:1 ratio. NMR studies on one of these duplexes brought evidence of the intercalation of the bisacridine at the position involving the propanediol linkage. These results suggest that this system could be used to target a specific reaction at a preselected position using the bisacridine derivative as carrier for the reactive species.

Acridines↗

PubMed1992

Triple-helix formation by oligonucleotides containing the three bases thymine, cytosine, and guanine.

A homopurine-homopyrimidine sequence of human immunodeficiency virus (HIV) proviral DNA was chosen as a target for triple-helix-forming oligonucleotides. An oligonucleotide containing three bases (thymine, cytosine, and guanine) was shown to bind to its target sequence under physiological conditions. This oligonucleotide is bound in a parallel orientation with respect to the homopurine sequence. Thymines recognize A.T base pairs to form T.A.T base triplets and guanines recognize a run of G.C base pairs to form G.G.C base triplets. A single 5-methylcytosine was shown to stabilize the triple helix when incorporated in a stretch of thymines; it recognizes a single G.C base pair in a run of A.T base pairs. These results provide some of the rules required for choosing the more appropriate oligonucleotide sequence to form a triple helix at a homopurine-homopyrimidine sequence of duplex DNA. A psoralen derivative attached to the oligonucleotide containing thymine, 5-methylcytosine, and guanine was shown to photoinduce cross-linking of the two DNA strands at the target sequence in a plasmid containing part of the HIV proviral DNA sequence. Triplex formation and cross-linking were monitored by inhibition of Dra I restriction enzyme cleavage. The present results provide a rational basis for the development of triplex-forming oligonucleotides targeted to specific sequences of the HIV provirus integrated in its host genome.

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PubMed1992

Drug targeting: synthesis and endocytosis of oligonucleotide-neoglycoprotein conjugates.

Inhibition of gene expression by antisense oligonucleotides is limited by their low ability to enter cells. Knowing that sugar binding receptors, also called membrane lectins, efficiently internalize neoglycoproteins bearing the relevant sugar, 6-phosphomannose, for instance, oligonucleotides--substituted on their 5'-end with either a fluorescent probe or a radioactive label on the one hand, and bearing a thiol function on their 3'-end, on the other hand,--were coupled onto 6-phosphomannosylated proteins via a disulfide bridge. The oligonucleotide bound to 6-phosphomannosylated serum albumin is much more efficiently internalized roughly 20 times than the free oligonucleotide. Although most of the oligonucleotides are associated with vesicular compartments, oligonucleotides after releasing from the carrier by reduction of the disulfide bridge may find their way to reach the cytosol and then lead to an increase in the efficiency of the oligonucleotides.

Animals↗

PubMed1992

Oligodeoxynucleotide-directed photo-induced cross-linking of HIV proviral DNA via triple-helix formation.

The HIV proviral genome contains two copies of a 16 bp homopurine.homopyrimidine sequence which overlaps the recognition and cleavage site of the Dra I restriction enzyme. Psoralen was attached to the 16-mer homopyrimidine oligonucleotide, d5'(TTTTCT-TTTCCCCCCT)3', which forms a triple helix with this HIV proviral sequence. Two plasmids, containing part of the HIV proviral DNA, with either one (pLTR) or two (pBT1) copies of the 16-bp homopurine.homopyrimidine sequence and either 4 or 14 Dra I cleavage sites, respectively, were used as substrates for the psoralen-oligonucleotide conjugate. Following UV irradiation the two strands of the DNA targeted sequence were cross-linked at the triplex-duplex junction. The psoralen-oligonucleotide conjugate selectively inhibited Dra I enzymatic cleavage at sites overlapping the two triple helix-forming sequences. A secondary triplex-forming site of 8 contiguous base pairs was observed on the pBT1 plasmid when binding of the 16 base-long oligonucleotide was allowed to take place at high oligonucleotide concentrations. Replacement of a stretch of six cytosines in the 16-mer oligomer by a stretch of six guanines increased binding to the primary sites and abolished binding to the secondary site under physiological conditions. These results demonstrate that oligonucleotides can be designed to selectively recognize and modify specific sequences in HIV proviral DNA.

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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.

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PubMed1992

Specific inhibition of transcription by triple helix-forming oligonucleotides.

Homopyrimidine oligonucleotides bind to the major groove of a complementary homopyrimidine.homopurine stretch by triple helix formation. The bla gene from transposon Tn3 contains a homopyrimidine.homopurine sequence of 13 base pairs located just downstream of the RNA polymerase binding site. A 13-mer homopyrimidine oligonucleotide targeted to this sequence was tested for its effect on transcription of the bla gene in vitro. We show that the consequence of triple helix formation in front of the Escherichia coli RNA polymerase-promoter complex is to block the holoenzyme at its start site during a period that is dependent on temperature. The temperature dependence of transcription inhibition shows a direct correlation between this effect and the stabilization of the triple helix. Substitution of 5-methylcytosine to cytosine in the 13-mer oligonucleotide enhances triplex stability and transcription inhibition. Transcription inhibition by this synthetic repressor was also confirmed by footprinting studies demonstrating its specificity of action. The 13-mer oligonucleotide containing a psoralen derivative covalently linked to its 5' end shows an irreversible and specific inhibition of transcription initiation after exposure to light of wavelength greater than 310 nm.

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PubMed1992

Mechanisms of the inhibition of reverse transcription by antisense oligonucleotides.

We have demonstrated that the synthesis of cDNA by avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases can be prevented by oligonucleotides bound to the RNA template approximately 100 nucleotides remote from the 3' end of the primer. The RNA was truncated at the level of the antisense oligonucleotide-RNA duplex during the reverse transcription. The key role played by the reverse transcriptase-associated RNase H activity in the inhibition process was shown by the use of (i) inhibitors of RNase H (NaF or dAMP), (ii) Moloney murine leukemia virus reverse transcriptase devoid of RNase H activity, or (iii) alpha-analogues of oligomers that do not elicit RNase H-catalyzed RNA degradation. In all three cases the inhibitory effect was either reduced (NaF, dAMP) or totally abolished. However, an alpha-oligomer bound to the sequence immediately adjacent to the primer-binding site prevented reverse transcription. Therefore, initiation of polymerization can be blocked by means of an RNase H-independent mechanism, whereas arrest of a growing cDNA strand can be achieved only by an oligonucleotide mediating cleavage of the template RNA.

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PubMed1991

The lac repressor and its N-terminal headpiece can bind a mini-operator containing a hairpin loop made of a hexaethylene glycol chain.

The binding of the lac repressor and the lac repressor N-terminal headpiece to a mini-operator with a hairpin loop made of a hexaethylene glycol chain was investigated using circular dichroism spectroscopy. The lac repressor's headpiece binds to the modified mini-operator with the same affinity as to a mini-operator of the same sequence without the hexaethylene glycol loop. The conformational effect due to the binding is not affected by the presence of the hexaethylene loop. It is also shown that the entire lac repressor binds to this modified mini-operator inducing a conformational change.

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PubMed1991

Synthesis and physicochemical properties of oligonucleotides built with either alpha-L or beta-L nucleotides units and covalently linked to an acridine derivative.

Modified deoxynucleosides 2'-deoxy-beta-L-uridine, beta-L-thymidine, alpha-L-thymidine, 2'-deoxy-beta-L-adenosine and 2'-deoxy-alpha-L-adenosine were synthesized and assembled as homooligomers, respectively: octa-beta-L-deoxyuridylates, octa beta-L and alpha-L-thymidylates and tetra beta-L and alpha-L-deoxyadenylates. These unnatural oligomers were then substituted with an acridine derivative. The binding studies of these modified oligonucleotides with D-ribo- and D-deoxyribopolynucleotides were carried out by absorption spectroscopy. While beta-L-d(Up)8m5Acr, beta-L-(Tp)8m5Acr, alpha-L-(Tp)8m5Acr did not interact with poly(rA) and poly(dA), beta-L-d(Ap)4m5Acr and alpha-L-d(Ap)4m5Acr did form double and triple helices with poly(rU) and poly(dT), respectively. Their stability towards nuclease digestion was studied through comparison with that of octa-beta-D-thymidylate and tetra beta-D-deoxyadenylate covalently linked to an acridine derivative. One endonuclease (nuclease P1 from Penicillium citrinum) and two exonucleases (a 3'-exonuclease from Crotalus durissus venom and a 5'-exonuclease extracted from calf thymus) were employed. beta-L- and alpha-L-oligomers demonstrate a high resistance toward nuclease digestion.

Acridines↗

PubMed1991

Site-specific intercalation at the triplex-duplex junction induces a conformational change which is detectable by hypersensitivity to diethylpyrocarbonate.

Using site-specific intercalation directed by intermolecular triplex formation, the conformation of an intercalation site in DNA was examined by footprinting with the purine-specific (A much greater than G) reagent diethylpyrocarbonate. Site specific intercalation was achieved by covalently linking an intercalator to the 5' end of a homopyrimidine oligodeoxynucleotide, which bound to a homopurinehomopyrimidine stretch in a recombinant plasmid via intermolecular triplex formation. This directs intercalation to a single site in 3kb of DNA at the 5' triplex-duplex junction. Footprinting with diethylpyrocarbonate and dimethylsulphate revealed strong protection from modification of adenine residues within the triple-helix in concordance with their Hoogsteen pairing with the third strand, and a strong hypersensitivity to diethylpyrocarbonate at the first adenine of the duplex. This result indicates that intercalation at this site induces a conformational change at the 5' triplex-duplex junction. Furthermore, the same diethlypyrocarbonate hypersensitivity was observed with an unmodified triple-strand forming oligonucleotide and a range of intercalating molecules present in solution. Thus the 5' triplex-duplex junction is a strong binding site for some intercalating molecules and the junction undergoes a conformational change which is sensitive to diethylpyrocarbonate upon insertion of the planar aromatic chromophore. This conformational change can be used to direct a single-strand cut in duplex DNA to a defined site.

Acridines↗

PubMed1991

Triple-helix formation by alpha oligodeoxynucleotides and alpha oligodeoxynucleotide-intercalator conjugates.

Base-pair sequences in double-stranded DNA can be recognized by homopyrimidine oligonucleotides that bind to the major groove at homopurine.homopyrimidine sequences thereby forming a local triple helix. To make oligodeoxynucleotides resistant to nucleases, we replaced the natural (beta) anomers of the nucleotide units by the synthetic (alpha) anomers. The 11-mer alpha oligodeoxynucleotide 5'-d(TCTCCTCCTTT)-3' binds to the major groove of DNA in an antiparallel orientation with respect to the homopurine strand, whereas a beta oligonucleotide adopts a parallel orientation. When an intercalating agent was attached to the 3' end of the alpha oligodeoxynucleotide, a strong stabilization of the triple helix was observed. A 16-base-pair homopurine.homopyrimidine sequence of human immunodeficiency virus proviral DNA was chosen as a target for a 16-mer homopyrimidine alpha oligodeoxynucleotide. A restriction enzyme that cleaves DNA at the junction of the homopurine.homopyrimidine sequence was inhibited by triple-helix formation. The 16-mer alpha oligodeoxynucleotide substituted by an intercalating agent was approximately 20 times more efficient than the unsubstituted oligomer. Nuclease-resistant alpha oligodeoxynucleotides offer additional possibilities to control gene expression at the DNA level.

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PubMed1991

Sequence-specific photo-induced cross-linking of the two strands of double-helical DNA by a psoralen covalently linked to a triple helix-forming oligonucleotide.

On the basis of the structure of DNA-psoralen bis adducts (formed by psoralen with two thymines on opposite strands), a psoralen-oligonucleotide conjugate was designed to photoinduce a cross-link between the two DNA strands at a specific sequence. Psoralen was attached via its C-5 position to a 5'-thiophosphate group of an 11-mer homopyrimidine oligonucleotide. The 11-mer binds to an 11-base-pair homopurine.homopyrimidine sequence of a DNA fragment, where it forms a triple helix. Upon near-UV-irradiation, the two strands of DNA are crosslinked at the TpA step present at the triplex-duplex junction. The reaction is specific for the homopurine.homopyrimidine DNA sequence and requires both oligonucleotide recognition of the DNA major groove and intercalation of psoralen at the triplex-duplex junction. The yield of the photo-induced cross-linking reaction is quite high (greater than 80%). Such psoralen-oligonucleotide conjugates are probes of sequence-specific triple-helix formation and could be used to selectively control gene expression or to induce site-directed mutations.

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PubMed1991

Inhibition of translation initiation by antisense oligonucleotides via an RNase-H independent mechanism.

We have used alpha-oligomers as antisense oligonucleotides complementary to three different sequences of the rabbit beta-globin mRNA: a region adjacent to the cap site, a region spanning the AUG initiation codon or a sequence in the coding region. These alpha-oligonucleotides were synthesized either with a free 5' OH group or linked to an acridine derivative. The effect of these oligonucleotides on mRNA translation was investigated in cell-free extracts and in Xenopus oocytes. In rabbit reticulocyte lysate and in wheat germ extracts oligomers targeted to the cap site and the initiation codon reduced beta-globin synthesis in a dose-dependent manner, whereas the target mRNA remained intact. The anti-cap alpha-oligomer was even more efficient that its beta-counterpart in rabbit reticulocyte lysate. In contrast, only the alpha-oligomer, linked to the acridine derivative, complementary to the cap region displayed significant antisense properties in Xenopus oocytes. Therefore initiation of translation can be arrested by oligonucleotide/RNA hybrids which are not substrates for RNase-H.

Animals↗

PubMed1991

Design of sequence-specific bifunctional nucleic acid ligands.

Homopyrimidine oligodeoxynucleotides have been covalently linked to intercalating agents. These bifunctional nucleic acid ligands bind to the major groove of DNA at homopurine.homopyrimidine sequences, where they form triple helices. The homopyrimidine oligonucleotide binds parallel to the purine strand of the double helix. Two hydrogen bonds are formed between bases of the oligonucleotide and the purines engaged in Watson-Crick base pairs. The intercalating agent inserts its aromatic ring at the triplex-duplex junction, resulting in a strong stabilization of the triple helical structure. Bifunctional oligonucleotide-intercalator conjugates provide new tools for a selective control of gene expression. In addition, irreversible reactions can be targeted to the oligonucleotide recognition sequence. Cleavage reactions can be induced by a copper-phenanthroline chelate or an ellipticine derivative covalently linked to the triple helix-forming oligonucleotide.

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PubMed1991

Short modified antisense oligonucleotides directed against Ha-ras point mutation induce selective cleavage of the mRNA and inhibit T24 cells proliferation.

We have used derivatized antisense oligodeoxynucleotides both in vitro and in vivo specifically to inhibit translation of the activated human oncogene Ha-ras. The oligonucleotides (5'-CCACACCGA-3') were targeted to a region of Ha-ras mRNA including the point mutation G----T at the 12th codon which leads to a Gly----Val substitution in the ras p21 protein. They were linked to an intercalating agent and/or to a hydrophobic tail, both to increase their affinity for their mRNA target and to enhance their uptake by tumor cells. A cell-free translation system was used to demonstrate an RNase H-dependent specific inhibition of activated ras protein synthesis. 50% inhibition was observed at a concentration of 0.5 microM of the most efficient oligonucleotide (5'-substitution with an acridine derivative and 3'-substitution by a dodecanol chain). This inhibitory effect stems from a point mutation-sensitive cleavage of the mRNA and it mirrors the growth inhibition obtained with T24 bladder carcinoma cells, which carry activated Ha-ras. The proliferation of HBL100 cells (non tumorigenic human mammary cell line) which carry two copies of normal Ha-ras was unaffected. This study shows that it is possible to design antisense agents that will inactivate the mutated oncogene but not the protooncogene which is generally essential to cell survival.

Animals↗

PubMed1991

Recognition and photo-induced cleavage and cross-linking of nucleic acids by oligonucleotides covalently linked to ellipticine.

Oligopyrimidines covalently linked to ellipticine derivatives form duplex and triplex structures with target single-stranded oligopurine sequences. They also bind to duplex DNA at homopurine-homopyrimidine sequences where they form local triple helices. Irradiation at wavelengths longer than 300 nm of the complex formed by an oligonucleotide-ellipticine conjugate with its target sequence induced (i) cleavage of the target at bases located in close proximity to the dye and (ii) cross-linking of the target sequence to the derivatized oligonucleotide. Both cross-linking and cleavage reactions decreased when temperature increased with a half-transition corresponding to the dissociation of the oligonucleotide-ellipticine conjugate from its target nucleic acid, demonstrating that the observed photochemical effects are dependent on hybrid formation. When the target was a double-stranded DNA, photochemical reactions were observed on both strands of the duplex. Photo-induced cross-linking was more efficient than cleavage when the target was single-stranded; the reverse was observed when the target was duplex DNA.

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PubMed1991

Design of bifunctional oligonucleotide intercalator conjugates as inhibitors of gene expression.

Oligonucleotide-intercalator conjugates have been designed to control gene expression at the translational and transcriptional level. The intercalator provides an additional binding energy when the oligonucleotide binds either to a complementary sequence on a single-stranded nucleic acid or to a homopurine.homopyrimidine sequence on duplex DNA. The oligonucleotide-intercalator conjugate can arrest translation of a mRNA (the "antisense" strategy); it can block transcription of DNA (the "antigene" strategy). Some of the intercalators that we have chosen can induce irreversible reactions in their target sequence. Here we summarize the reactions that can be targeted to specific sequences of duplex DNA. Phenanthroline induces cleavage of the two strands of duplex DNA in the presence of Cu(II) and a reducing agent. Ellipticine derivatives can be used to photo-induce cleavage. Psoralen derivatives can cross-link the two strands of DNA under near UV irradiation. In all cases the chemical or photochemical reactions are targeted to a specific sequence of duplex DNA.

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