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J J Toulmé

Publications and source records attributed to J J Toulmé.

At least 19 recordsLinked to original sources
PubMed2001

DNA aptamers selected against the HIV-1 RNase H display in vitro antiviral activity.

The DNA polymerase of the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a target widely used to inhibit HIV-1 replication. In contrast, very few inhibitors of the RNase H activity associated with RT have been described, despite the crucial role played by this activity in viral proliferation. DNA ligands with a high affinity for the RNase H domain of HIV-1 RT were isolated by systematic evolution of ligands by an exponential enrichment strategy (SELEX), using recombinant RTs with or without the RNase H domain. The selected oligonucleotides (ODNs) were able to inhibit in vitro the HIV-1 RNase H activity, while no effect was observed on cellular RNase H. We focused our interest on two G-rich inhibitory oligonucleotides. Model studies of the secondary structure of these ODNs strongly suggested that they were able to form G-quartets. In addition to the inhibition of HIV-1 RNase H observed in a cell free system, these ODNs were able to strongly diminish the infectivity of HIV-1 in human infected cells. Oligonucleotides described here may serve as leading compounds for the development of specific inhibitors of this key retroviral enzyme activity.

Anti-HIV Agents↗
PubMed2001

Modulation of RNA function by oligonucleotides recognizing RNA structure.

Numerous RNA structures are responsible for regulatory processes either because they constitute a signal, like the hairpins or pseudoknots involved in ribosomal frameshifting, or because they are binding sites for proteins such as the trans-activating responsive RNA element of the human immunodeficiency virus whose binding to the viral protein Tat and cellular proteins allows full-length transcription of the retroviral genome. Selective ligands able to bind with high affinity to such RNA motifs may serve as tools for dissecting the molecular mechanisms in which they are involved. Such ligands might also constitute prototypes of therapeutic agents when RNA structures play a role in the expression of dysfunctional genes or in the multiplication of pathogens. Different classes of ligands (aminoglycosides, interacalating agents, peptides) are of interest to this aim. However, oligonucleotides deserve particular consideration. They have been extensively used in the frame of the antisense strategy. The apparent simplicity of this rational approach is, at first sight, very attractive. Indeed, numerous successful studies have been published describing the efficient inhibition of translation, splicing, or reverse transcription in cell-free systems, in cultured cells, or in vivo by oligomers complementary to an RNA region. However, RNA structures restrict the access of the target site to the antisense sequence: The competition between the intramolecular association of RNA regions weakens or even abolishes the antisense effect. Various possibilities have been developed to circumvent this limitation. This includes both rational and combinatorial strategies. High-affinity oligomers were designed to invade the RNA structure. Alternatively, triplex-forming oligonucleotides (TFO) and aptamers may recognize the folded RNA motif. Whereas the use of TFOs is rather limited owing to the strong sequence constraints for triple-helix formation, in vitro selection offers a way to explore vast oligoribo or oligodeoxyribo libraries to identify strong, selective oligonucleotide binders. The candidates (aptamers) selected against the TAR RNA element of HIV-1, which form stable loop-loop (kissing) complexes with the target, provide interesting examples of oligonucleotides recognizing a functional RNA structure through an important contribution of tertiary interactions.

Animals↗
PubMed2001

A method to select chemically modified aptamers directly.

In vitro selection is a strategy to identify high-affinity ligands of a predetermined target among a large pool of randomized oligonucleotides. Most in vitro selections are performed with unmodified RNA or DNA sequences, leading to ligands of high affinity and specificity (aptamers) but of very short lifetime in the ex vivo and in vivo context. Only a very limited number of modified triphosphate nucleotides conferring nuclease resistance to the oligomer can be incorporated by polymerases. This encourages the development of alternative methods for the identification of nuclease-resistant aptamers. In this paper, we describe such a method. After selection of 2'O-methyl oligonucleotides against the TAR RNA structure of HIV-1, the complementary DNA sequences are fished out of a pool of randomized oligodeoxynucleotides by Watson-Crick hybridization. The DNA-fished sequences are amplified by PCR as double and single strands, the latter being used to fish back the chemically modified candidates from the initial library. This procedure allows an indirect amplification of the selected candidates. This enriched pool of modified sequences is then used for the next selection round against the target.

Base Sequence↗
PubMed2000

NMR characterization of a kissing complex formed between the TAR RNA element of HIV-1 and a DNA aptamer.

This work presents the first structural analysis of an RNA-DNA complex consisting of an 18 nt RNA hairpin and a 20 nt DNA aptamer. The DNA molecule was previously selected, from a randomly synthesized library, against the transactivation response element (TAR) involved in transcriptional regulation of the HIV genome. The DNA aptamer used in the present study is an imperfect stem-loop with the sequence 5'-ACTCCCAT-3', characteristic of the selected candidates, in the apical loop. This octameric motif contains five bases complementary to the TAR loop sequence 5'-CUGGGA-3'. The use of homo- and heteronuclear NMR spectroscopy allowed assignment of the complex resonances and resolution of its secondary structure. Evidence is given for a kissing complex fold, which consists of a quasi-continuous helix formed by one stem of DNA, one stem of RNA and a central hybrid helix comprising 5 bp. Two out of helices residues of DNA and one of RNA connect the DNA-RNA loop-loop helix to the stem of either partner in the complex. In addition, two thymines of the DNA stem are engaged in a non-canonical T.T base pair.

Base Pairing↗
PubMed2000

4-amino-1H-benzo[g]quinazoline-2-one: a fluorescent analog of cytosine to probe protonation sites in triplex forming oligonucleotides.

We developed a new fluorescent analog of cytosine, the 4-amino-1H-benzo[g]quinazoline-2-one, which constitute a probe sensitive to pH. The 2'-O-Me ribonucleoside derivative of this heterocycle was synthesized and exhibited a fluorescence emission centered at 456 nm, characterized by four major excitation maxima (250, 300, 320 and 370 nm) and a fluorescence quantum yield of Phi = 0.62 at pH 7.1. The fluorescence emission maximum shifted from 456 to 492 nm when pH was decreased from 7.1 to 2.1. The pK(a) (4) was close to that of cytosine (4.17). When introduced in triplex forming oligonucleotides this new nucleoside can be used to reveal the protonation state of triplets in triple-stranded structures. Complex formation was detected by a significant quenching of fluorescence emission (approximately 88%) and the N-3 protonation of the quinazoline ring by a shift of the emission maximum from 485 to 465 nm. Using this probe we unambiguously showed that triplex formation of the pyrimidine motif does not require the protonation of all 4-amino-2-one pyrimidine rings.

Base Sequence↗
PubMed2000

Inhibition of in vitro and ex vivo translation by a transplatin-modified oligo(2'-O-methylribonucleotide) directed against the HIV-1 gag-pol frameshift signal.

A 2'-O-methylribooligonucleotide containing a G1.U.G3 triad modified by trans-diamminedichloro-platinum(II) was targeted to the RNA region responsible for the gag-pol frameshifting during translation of the HIV-1 mRNA. The binding of the platinated oligonucleotide to its target RNA induced a rearrangement of the (G1, G3)-intrastrand crosslink, leading to the formation of an intermolecular oligonucleotide-RNA G-A crosslink. This resulted in the selective arrest of translation of a luciferase gene placed downstream of the HIV-1 frameshift signal both in a cell-free extract (rabbit reticulocyte lysate) and in RNA-transfected cells. A specific inhibition of luciferase activity was still observed when the oligonucleotide-RNA complex was not pre-formed prior to either translation or transfection. Moreover, a selective inhibition was also observed when the oligonucleotide and the plasmid DNA encoding the luciferase and bearing the RNA gag- pol frameshifting signal were co-transfected in NIH 3T3 cultured cells. Therefore the intra-strand-->interstrand conversion of the platinum crosslink kinetically competes with the translation machinery and blocks the polypeptide elongation. These transplatin-modified oligonucleotides which operate within a live cell on a 'real-time' basis and do not need an external triggering signal constitute a promising new class of selective reactive probes.

3T3 Cells↗
PubMed2000

Towards the selection of phosphorothioate aptamers optimizing in vitro selection steps with phosphorothioate nucleotides.

The high affinity of a given nucleic acid for a protein ligand can be used to isolate specific inhibitors of enzymes involved in pathological situations. The latter property is the basis of the SELEX (systematic evolution of ligands by exponential enrichment) technique. Recently, several potent nucleic acids inhibitors of HIV-1 replication have been isolated using the SELEX approach. However, phosphodiester oligodeoxynucleotides (PO-ODNs) were not used as antiviral agents because of their sensitivity to nucleases. Our goal in this work was to explore the possibility of selecting, from a fully substituted phosphorothioate library, oligonucleotides having both a strong affinity for HIV-1 reverse transcriptase (RT) and nuclease resistance. HIV-1 RT initiates in vivo reverse transcription from the 3' end of a host tRNALys. Although phosphorothioate ODNs (PS-ODNs) have been claimed to bind unspecifically to proteins, we have shown previously that an ODN corresponding to the acceptor stem of tRNALys was able to inhibit specifically HIV-1 replication in HIV-1 infected cells, without showing cytotoxicity up to 10 microM. As the SELEX strategy requires 'in vitro' transcription and reverse transcription of the selected DNA, we have assayed the available PS precursors as a model system by using PS-dNTPs and rNTPs. We have also developed an experimental procedure to optimize the incorporation of four PS-dNTPs during the PCR step of the SELEX approach. In the course of this work, we have showed that the PS-dGTP is a strong inhibitor of thermostable DNA polymerases as well as of HIV-1 RT.

Base Sequence↗
PubMed2000

Aptamers: selected oligonucleotides for therapy.

In vitro selection constitutes a unique way to explore vast libraries (up to 10(14) to 10(15) different molecules) of randomly synthesized nucleic acids. Selected RNA or DNA sequences (aptamers) may help to understand the molecular interactions and processes of interest for human diseases. Aptamers can be chemically-modified for improved efficiency and use in complex biological media. Moreover, aptamers selected in vitro may retain their activity in vivo and thus offer novel perspectives for gene therapy and the design of new drugs.

Animals↗
PubMed1999

DNA aptamers selected against the HIV-1 trans-activation-responsive RNA element form RNA-DNA kissing complexes.

In vitro selection was performed in a DNA library, made of oligonucleotides with a 30-nucleotide random sequence, to identify ligands of the human immunodeficiency virus type-1 trans-activation-responsive (TAR) RNA element. Aptamers, extracted after 15 rounds of selection-amplification, either from a classical library of sequences or from virtual combinatorial libraries, displayed an imperfect stem-loop structure and presented a consensus motif 5'ACTCCCAT in the apical loop. The six central bases of the consensus were complementary to the TAR apical region, giving rise to the formation of RNA-DNA kissing complexes, without disrupting the secondary structure of TAR. The RNA-DNA kissing complex was a poor substrate for Escherichia coli RNase H, likely due to steric and conformational constraints of the DNA/RNA heteroduplex. 2'-O-Methyl derivatives of a selected aptamer were binders of lower efficiency than the parent aptamer in contrast to regular sense/antisense hybrids, indicating that the RNA/DNA loop-loop region adopted a non-canonical heteroduplex structure. These results, which allowed the identification of a new type of complex, DNA-RNA kissing complex, demonstrate the interest of in vitro selection for identifying non-antisense oligonucleotide ligands of RNA structures that are of potential value for artificially modulating gene expression.

Base Sequence↗
PubMed1999

Binding of oligopyrimidines to the RNA hairpin responsible for the ribosome gag-pol frameshift in HIV-1.

The 12 bp stem of the RNA hairpin responsible for the gag-pol frameshifting of the ribosomes during translation of the polycistronic HIV-1 mRNA has a pyrimidine-rich 5' strand and, consequently, a purine-rich 3' strand. Electrophoretic mobility shift assays have shown that DNA oligopyrimidines, 12 and 20 nucleotides long (but not oligopurines or G,T-containing oligomers), designed to form triplexes actually bind to the double-stranded RNA target. RNase V1 footprinting studies have confirmed the interaction between the hairpin stem and the RNA and 2'-O-methyl oligoribonucleotide analogues of the 12-mer oligodeoxypyrimidine as well as 5 propynyl,cytosine, containing the 12-mer oligodeoxypyrimidine, bind more strongly to the RNA target than the unmodified parent DNA oligomer. The complexes formed by the RNA hairpin and either the 12-mer oligodeoxypyrimidine or the 20-mer oligopyrimidine are stable at a neutral pH and in the absence of Mg2+ but blocked neither the reverse transcription nor cell-free translation of a RNA template in which the gag-pol frameshifting hairpin was inserted at the 5' end of the luciferase open reading frame.

Biological Assay↗
PubMed1999

Antisense oligonucleotides containing modified bases inhibit in vitro translation of Leishmania amazonensis mRNAs by invading the mini-exon hairpin.

Complementary oligodeoxynucleotides (ODNs) that contain 2-aminoadenine and 2-thiothymine interact weakly with each other but form stable hybrids with unmodified complements. These selectively binding complementary (SBC) agents can invade duplex DNA and hybridize to each strand (Kutyavin, I. V., Rhinehart, R. L., Lukhtanov, E. A., Gorn, V. V., Meyer, R. B., and Gamper, H. B. (1996) Biochemistry 35, 11170-11176). Antisense ODNs with similar properties should be less encumbered by RNA secondary structure. Here we show that SBC ODNs strand invade a hairpin in the mini-exon RNA of Leishmania amazonensis and that the resulting heteroduplexes are substrates for Escherichia coli RNase H. SBC ODNs either with phosphodiester or phosphorothioate backbones form more stable hybrids with RNA than normal base (NB) ODNs. Optimal binding was observed when the entire hairpin sequence was targeted. Translation of L. amazonensis mRNA in a cell-free extract was more efficiently inhibited by SBC ODNs complementary to the mini-exon hairpin than by the corresponding NB ODNs. Nonspecific protein binding in the cell-free extract by phosphorothioate SBC ODNs rendered them ineffective as antisense agents in vitro. SBC phosphorothioate ODNs displayed a modest but significant improvement of leishmanicidal properties compared with NB phosphorothioate ODNs.

Animals↗
PubMed1999

In vitro selection identifies key determinants for loop-loop interactions: RNA aptamers selective for the TAR RNA element of HIV-1.

We selected RNA aptamers specific for the trans-activation responsive (TAR) RNA, a stem-loop structure crucial for the transcription of the integrated genome of the human immunodeficiency virus. Most of the selected sequences could be folded as imperfect hairpins and displayed a 5'-GUCCCAGA-3' consensus motif constituting the apical loop. The six central bases of this consensus sequence are complementary to the entire TAR loop, leading to the formation of TAR RNA-aptamer "kissing" complexes. The consensus G and A residues closing the aptamer loop contributed to the high affinity (Kd = 30 nM at 23 degrees C) of the aptamers for the TAR RNA. This G A pair was shown to be crucial for binding to TAR at a low magnesium concentration. The selection also identified 5'-PuPy and 5'-PyPu base pairs at alpha and beta positions of the stem, next to the loop, respectively. This strategy offered a way to identify key determinants of loop-loop interactions and to generate high affinity ligands of TAR RNA structure.

Base Pairing↗
PubMed1998

Modified (PNA, 2'-O-methyl and phosphoramidate) anti-TAR antisense oligonucleotides as strong and specific inhibitors of in vitro HIV-1 reverse transcription.

Natural beta-phosphodiester 16mer and 15mer antisense oligonucleotides targeted against the HIV-1 and HIV-2 TAR RNAs respectively were previously described as sequence-specific inhibitors of in vitro retroviral reverse transcription. In this work, we tested chemically modified oligonucleotide analogues: alpha-phosphodiester, phosphorothioate, methylphosphonate, peptide nucleic acid or PNA, 2'- o -methyl and (N3'-P5') phosphoramidate versions of the 16mer anti-TAR oligonucleotide. PNA, 2'- O -methyl and (N3'-P5') phosphoramidate oligomers showed a strong inhibitory effect compared with the unmodified 16mer, with reverse transcription inhibition (IC50) values in the nanomolar range. The inhibition was sequence-specific, as scrambled and mismatched control oligonucleotides were not able to inhibit cDNA synthesis. No direct binding of the 2'- O -methyl, PNA or (N3'-P5') phosphoramidate anti-TAR oligonucleotides to the HIV-1 reverse transcriptase was observed. The higher T m obtained with 2'- O -methyl, (N3'-P5') phosphoramidate and PNA molecules concerning the annealing with the stem-loop structure of the TAR RNA, in comparison with the beta-phosphodiester oligonucleotides, is correlated with their high inhibitory effect on reverse transcription.

Anti-HIV Agents↗
PubMed1998

Benzoquinazoline derivatives as substitutes for thymine in nucleic acid complexes. Use of fluorescence emission of benzo[g]quinazoline-2,4-(1H,3H)-dione in probing duplex and triplex formation.

Triple helix formation obeys structural features that do not allow accommodation of every double-stranded sequence; it requires the occurrence of homopurine stretches. A further constraint comes from the weak energy of interaction between the third strand and the double-stranded target. In an attempt to design bases leading to increased stability of triplexes, we explored the ability of modified bases with an extended aromatic domain to increase third strand binding through stacking interactions. We report here the use of benzo[g]- and benzo[f]quinazoline-2,4-dione-(1H,3H)-dione as substitutes for thymine in the canonical TAT triplet. The synthesis and characterization of the beta nucleoside derivatives of benzoquinazolines are described. Triplex-forming oligonucleotides containing these modified bases have been prepared, and their ability to form triplexes has been evaluated by UV absorption-monitored thermal denaturation measurements. Benzo[g]quinazoline and benzo[f]quinazoline formed triple-stranded structures with slightly decreased stabilities. In addition, benzo[g]quinazoline revealed strong fluorescence emission properties which can be used to monitor selectively the formation of triple-helical structures. Annealing of benzo[g]quinazoline to complementary strands did not produce any fluorescence modification. But when it was introduced into the Hoogsteen strand of PyPuPy complexes, the fluorescence intensity was reduced and the emission maximum was shifted to short wavelengths.

DNA↗
PubMed1998

Mapping of a minimal AU-rich sequence required for lipopolysaccharide-induced binding of a 55-kDa protein on tumor necrosis factor-alpha mRNA.

In monocyte/macrophage cells, the translation of tumor necrosis factor-alpha (TNF-alpha) mRNA is tightly controlled. In unstimulated cells, TNF-alpha mRNA is translationally repressed. However, upon stimulation of the cells with various agents (e.g. lipopolysaccharides (LPS) and viruses), this repression is overcome and translation occurs. The key element in this regulation is the AU-rich sequence present in the 3'-untranslated region of TNF-alpha mRNA. Several groups have described the binding of proteins on AU-rich elements (AREs). We have previously reported the binding of two cytosolic protein complexes (1 and 2) to the TNF-alpha mRNA ARE, one of which (complex 2) is observed only following induction of TNF-alpha production by LPS. In this report, we have demonstrated that complex 1 involves a long fragment of the ARE, whereas the formation of the LPS-inducible complex 2 requires a minimal sequence which corresponds to the nonanucleotide UUAUUUAUU. Furthermore, we show that the RNA-binding protein involved in complex 2 has an apparent molecular mass of 55 kDa. Finally, we tested other AREs for their ability to form complex 2. We observed that the ARE derived from granulocyte/macrophage colony-stimulating factor mRNA, which does contain the nonanucleotide, is able to sustain the LPS-induced binding of the 55-kDa protein. However, c-myc mRNA, which does not contain the nonanucleotide, is unable to promote the formation of any LPS-induced complex.

Animals↗