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C Giovannangeli

Publications and source records attributed to C Giovannangeli.

At least 19 recordsLinked to original sources
PubMed2001

Transcription inhibition induced by modified triple helix-forming oligonucleotides: a quantitative assay for evaluation in cells.

Oligonucleotides can bind to double-stranded DNA in a sequence-specific manner to form triple helices. Uniformly modified, pyrimidine-rich oligodeoxyribonuclotides containing internucleosidic N3'-P5' phosphoramidate linkages are known to form very stable triplexes with their DNA target. Psoralen-conjugated triple helix-forming oligonucleotides (Pso-TFOs) can additionally be photo-induced to become irreversibly bound to their targeted DNA sequence. Here, we have examined the ability of various 15-mer phosphoramidate TFOs targeted to the HIV-1 polypurine tract (PPT) sequence to prevent transcription elongation in cell cultures; the PPT sequence has been cloned in the transcribed region of a reporter firefly luciferase gene (luc) and transient expression experiments performed. We show that the level of transcription inhibition of the reporter gene in cells perfectly correlates with the amount of covalent triplex at the PPT site. The efficacy of non-covalent triplexes (either omitting the irradiation step with the psoralen conjugate, or using the unsubstituted oligonucleotide) has been studied in our expression system; the oligonucleotides were introduced into living cells by cationic lipid-mediated delivery or directly into the cell nucleus by microinjection. This experimental approach allowed us to evaluate the intrinsic activity of triplexes as transcriptional inhibitors; transcription elongation was inhibited in cells in a sequence-dependent and concentration-dependent manner. This experimental system is convenient for quantitative and fast evaluation of new chemistries of antigene oligonucleotides as inhibitors of gene expression in cells and in vivo.

Base Sequence↗
PubMed2001

Synthesis and properties of triple helix-forming oligodeoxyribonucleotides containing 7-chloro-7-deaza-2'-deoxyguanosine.

Multiple incorporations of 7-chloro-7-deaza-2'-deoxyguanosine in place of 2'-deoxyguanosine have been performed into a triple helix-forming oligodeoxyribonucleotide involving a run of six contiguous guanines designed to bind in a parallel orientation relative to the purine strand of the DNA target. The ability of these modified oligodeoxyribonucleotides to form triple helices in a buffer containing monovalent cations was studied by UV--melting curves analysis, gel shift assay and restriction enzyme protection assay. In the presence of Na(+), the incorporation of two, three or five modified nucleosides in the third strand has improved the efficacy of formation of the triplex as compared to that formed with the unmodified oligonucleotide. The stabilities of the three modified triplexes were similar. The coupling of 6-chloro-2-methoxy-9-(omega-hexylamino)-acridine to the 5'-end of the oligonucleotides containing modified nucleosides led to an increase in triplex stability similar to that observed when the acridine was added to the 5'-end of the unmodified oligonucleotide. In the presence of K(+), only the oligodeoxyribonucleotides containing modified G retained the ability to form triple helices with the same efficiency. The incorporation of the modified nucleoside has two effects: (i) it decreases TFO self-association, and (ii) it slightly increases triplex stability. The enhanced ability of the modified oligonucleotides containing 7-chloro-7-deaza-2'-deoxyguanosine over the parent oligomer to form triple helices was confirmed by inhibition of restriction enzyme cleavage using a circular plasmid containing the target sequence.

DNA, Viral↗
PubMed2001

Phosphoramidate oligonucleotides as potent antisense molecules in cells and in vivo.

Antisense oligonucleotides are designed to specifically hybridize to a target messenger RNA (mRNA) and interfere with the synthesis of the encoded protein. Uniformly modified oligonucleotides containing N3'-P5' phosphoramidate linkages exhibit (NP) extremely high-affinity binding to single-stranded RNA, do not induce RNase H activity, and are resistant to cellular nucleases. In the present work, we demonstrate that phosphoramidate oligonucleotides are effective at inhibiting gene expression at the mRNA level, by binding to their complementary target present in the 5'-untranslated region. Their mechanism of action was demonstrated by comparative analysis of three expression systems that differ only by the composition of the oligonucleotide target sequence (HIV-1 polypurine tract or PPT sequence) present just upstream from the AUG codon of the firefly luciferase reporter gene: the experiments have been done on isolated cells using oligonucleotide delivery mediated by cationic molecules or streptolysin O (SLO), and in vivo by oligonucleotide electrotransfer to skeletal muscle. In our experimental system phosphoramidate oligonucleotides act as potent and specific antisense agents by steric blocking of translation initiation; they may prove useful to modulate RNA metabolism while maintaining RNA integrity.

5' Untranslated Regions↗
PubMed2001

Site-directed inhibition of DNA replication by triple helix formation.

Sequence-specific DNA recognition can be achieved by the use of triplex-forming molecules, namely, oligonucleotides (TFO) and peptide nucleic acids (PNAs). They have been used to regulate transcription or induce genomic DNA modifications at a selected site in cells and, recently, in vivo. We have determined the conditions under which a triplex structure can inhibit DNA replication in cells. An oligopyrimidine.oligopurine sequence suitable for triplex formation was inserted in a plasmid on both sides of the SV40 origin of replication. This insert-containing plasmid was replicated in COS-1 cells together with the parent plasmid, and the ratio between the corresponding replicated DNAs was quantitated. Selective inhibition of replication of the insert-containing plasmid can be ascribed to ligand binding to the oligopyrimidine.oligopurine sequence. Inhibition of DNA replication was observed using triplex-forming molecules that induce either covalent binding at the double-stranded target sequence (with TFO-psoralen conjugate and irradiation) or noncovalent triplex formation after strand displacement (with bis-PNA). In contrast, in the absence of covalent cross-linking, TFOs (which have been shown to arrest transcription elongation) did not act on replication. These results open new perspectives for future design and use of specific inhibitors of intracellular DNA information processing.

Animals↗
PubMed2000

Targeted inhibition of transcription elongation in cells mediated by triplex-forming oligonucleotides.

Triple-helix-forming oligonucleotides (TFOs) bind in the major groove of double-stranded DNA at oligopyrimidine small middle dotoligopurine sequences and therefore are candidate molecules for artificial gene regulation, in vitro and in vivo. We recently have described oligonucleotide analogues containing N3'-P5' phosphoramidate (np) linkages that exhibited efficient inhibition of transcription elongation in vitro. In the present work we provide conclusive evidence that np-modified TFOs targeted to the HIV-1 polypurine tract (PPT) sequence can inhibit transcriptional elongation in cells, either in transient or stable expression systems. The same constructs were used in transient expression assays (target sequence on transfected plasmid) and in the generation of stable cell lines (target sequence integrated into cellular chromosomes). In both cases the only distinguishable feature between the cellular systems is the presence of an insert containing the wild-type PPT/HIV-1 sequence, a mutated version with two mismatches, or the absence of the insert altogether. The inhibitory action induced by np-TFOs was restricted to the cellular systems containing the complementary wild-type PPT/HIV-1 target, and consequently can be attributed only to a triple-helix-mediated mechanism. As a part of this study we also have applied an imaging technique to quantitatively investigate the dynamics of TFO-mediated specific gene silencing in single cells.

Animals↗
PubMed2000

Unambiguous demonstration of triple-helix-directed gene modification.

Triple-helix-forming oligonucleotides (TFOs), which can potentially modify target genes irreversibly, represent promising tools for antiviral therapies. However, their effectiveness on endogenous genes has yet to be unambiguously demonstrated. To monitor endogenous gene modification by TFOs in a yeast model, we inactivated an auxotrophic marker gene by inserting target sequences of interest into its coding region. The genetically engineered yeast cells then were treated with psoralen-linked TFOs followed by UV irradiation, thus generating highly mutagenic covalent crosslinks at the target site whose repair could restore gene function; the number of revertants and spectrum of mutations generated were quantified. Results showed that a phosphoramidate TFO indeed reaches its target sequence, forms crosslinks, and generates mutations at the expected site via a triplex-mediated mechanism: (i) under identical conditions, no mutations were generated by the same TFO at two other loci in the target strain, nor in an isogenic control strain carrying a modified target sequence incapable of supporting triple-helix formation; (ii) for a given target sequence, whether the triplex was formed in vivo on an endogenous gene or in vitro on an exogenous plasmid, the nature of the mutations generated was identical, and consistent with the repair of a psoralen crosslink at the target site. Although the mutation efficiency was probably too low for therapeutic applications, our results confirm the validity of the triple-helix approach and provide a means of evaluating the effectiveness of new chemically modified TFOs and analogs.

Base Sequence↗
PubMed2000

Psoralen adducts induced by triplex-forming oligonucleotides are refractory to repair in HeLa cells.

The use of triple helix-forming oligonucleotides constitutes an attractive strategy to regulate gene expression by inhibition of transcription. Psoralen-oligonucleotide conjugates form, upon irradiation, covalent triplexes and thereby modify the specific target sequence. The processing of such photoproducts on the promoter of the gene coding for the interleukin-2 receptor alpha chain was investigated in HeLa cells and HeLa nuclear extracts. We demonstrate that psoralen cross-links are not repaired within the cell extracts nor inside cells. The mechanism of repair inhibition was elucidated in vitro: the presence of the third strand oligonucleotide inhibits the incision step of the damaged target by repair endonucleases. These results demonstrate the possibility of using this approach to induce a persistent intracellular DNA damage at a specific site and to afford prolonged transcription inhibition.

Base Sequence↗
PubMed2000

Triplex-forming molecules for modulation of DNA information processing.

The design of specific DNA ligands is an important challenge in biological and biomedical sciences. Targeting the source of genetic information may allow highly efficient gene-directed modulation of cell function. Triplex-forming molecules specifically recognize oligopyrimidine/oligopurine sequences by hydrogen bonding interactions. In cell cultures they have been used successfully to downregulate or upregulate transcription in a gene-specific manner or to induce site-directed mutagenesis and recombination. For biotechnological applications, triplex-forming molecules are powerful tools for gene-specific chemistry.

Animals↗
PubMed1999

Covalent crosslinks introduced via a triple helix-forming oligonucleotide coupled to psoralen are inefficiently repaired.

Triple helix-forming oligonucleotides (TFOs) represent potentially powerful tools to artificially modulate gene activity. In particular, they can be used to specifically introduce a lesion into a selected target sequence: interstrand crosslinks and monoadducts can be introduced via TFOs coupled to psoralen. The efficiency of these strategies depends on the cell ability to repair these lesions, an issue which is still controversial. Here we show, using psoralen-coupled TFOs and the yeast as a convenient cellular test system, that interstrand crosslinks are quantitatively poorly repaired, resulting in an efficient modification of target gene activity. In addition, these lesions result in the introduction of mutations in a high proportion of cells. We show that these mutations are generated by the Error-Prone Repair pathway, alone or in combination with Nucleotide Excision Repair. Taken together, these results suggest that TFOs coupled to psoralen could be used to inactivate a gene with significant efficiency.

Adenosine Triphosphatases↗
PubMed1997

Accessibility of nuclear DNA to triplex-forming oligonucleotides: the integrated HIV-1 provirus as a target.

The control of gene transcription by antigene oligonucleotides rests upon the specific recognition of double-helical DNA by triplex-forming oligonucleotides. The development of the antigene strategy requires access to the targeted DNA sequence within the chromatin structure of the cell nucleus. In this sudy we have used HIV-1 chronically infected cells containing the HIV provirus as endogenous genes to demonstrate that the integrated HIV-1 proviral genome is accessible to triplex-forming oligonucleotides within cell nuclei. An oligonucleotide-psoralen conjugate targeted to the polypurine tract (PPT) of the HIV-1 proviral sequence was used as a tool to convert the noncovalent triple-helical complex into a covalent lesion on genomic DNA after UV irradiation of cells. Triplex-derived adducts were analyzed using two different methods. The photo-induced psoralen cross-link prevented cleavage of the target sequence by DraI restriction endonuclease, and the sequence-specific inhibition of cleavage was revealed and quantitated by Southern blot analysis. A quantitative analysis of cross-linking efficiency was also carried out by a competitive PCR-based assay. These two approaches allowed us to demonstrate that a triplex-forming oligonucleotide can recognize and bind specifically to a 15-bp sequence within the chromatin structure of cell nuclei.

Cell Membrane Permeability↗
PubMed1997

Sequence-specific control of gene expression by antigene and clamp oligonucleotides.

Control of gene expression at the transcriptional level can be achieved with triplex-forming oligonucleotides provided that the target sequence is accessible within the chromatin structure of cell nuclei. Using oligonucleotide-psoralen conjugates as probes we have shown that the promoter region of the gene encoding the alpha subunit of the interleukin 2 receptor and the polypurine tract of integrated HIV provirus can form sequence-specific, triple-helical complexes in cell cultures. Oligonucleotide-intercalator conjugates can inhibit transcription initiation by competing with transcription factor binding. Oligonucleotide analogues containing N3'-->P5' phosporamidate linkages form stable triple helices that are able to arrest transcription at the elongation step. A triple helix can also be formed on a single-stranded target by clamp oligonucleotides. A clamp targeted to the polypurine tract of HIV RNA is able to block reverse transcription of the viral RNA.

Animals↗
PubMed1997

Progress in developments of triplex-based strategies.

Recognition of B-DNA by oligonucleotides that form triple helices is a unique method to specifically recognize sequences of double-stranded DNA. Recently, some significant limitations of the triple-based applications have been overcome. Stable intermolecular triplexes can be formed under physiologic conditions. Binding affinities of modified oligonucleotides to their target sequence due to Hoogsteen or reverse Hoogsteen hydrogen bonding interactions are now in the range of those obtained for duplex formation via Watson-Crick hydrogen bonding interactions even if the kinetics may be quite different. Progress has been made toward developing general procedures to determine the molecular mechanisms of action of triplex-forming oligonucleotides (TFO) administered to cultured cells to provide a rational proof-of-concept for antigene strategies. The antigene strategy has reached a point where TFOs can be used to interfere with several biologic progresses (replication, transcription, recombination, repair) in relevant systems both in vitro and ex vivo.

Animals↗
PubMed1996

Efficient inhibition of transcription elongation in vitro by oligonucleotide phosphoramidates targeted to proviral HIV DNA.

Triplex-forming oligophosphoramidates containing thymines and cytosines or 5-methyl cytosines (5' T4CT4C6T 3') bind strongly to a 16 basepair oligopurine.oligopyrimidine sequence of HIV proviral DNA even at neutral pH. These triple-helical complexes formed with oligonucleotide analogues with N3'-->P5' phosphoramidate linkages are remarkably stable compared to oligonucleotides with natural phosphodiester linkages. In transcription assays the (T,C)-containing phosphoramidate oligomers induce an efficient arrest of both bacteriophage and eukaryotic transcriptional machineries under conditions where the isosequential phosphodiesters have no inhibitory effect. In both cases the RNA polymerase (SP6, T7 or Pol II) is physically blocked by the non-covalent triplex and RNA synthesis is stopped at the triplex site. However the eukaryotic transcription machinery is blocked more efficiently (at submicromolar concentration) than the bacteriophage polymerases. The analysis of the 3'-ends of the truncated transcripts provides evidence for differences in the termination patterns induced by the triplex barrier for the bacteriophage and the eukaryotic systems. This in vitro comparative study provides the basis for the rational design of strong transcriptional inhibitors. The efficient in vitro inhibition obtained using the phosphoramidate oligomers in the eukaryotic transcription assay makes them good candidates for the development of sequence-specific antigene agents.

Bacteriophages↗
PubMed1996

Specific inhibition of in vitro transcription elongation by triplex-forming oligonucleotide-intercalator conjugates targeted to HIV proviral DNA.

A 16-base pair oligo(purine)-oligo(pyrimidine) sequence present in the coding region of two HIV 1 proviral genes (pol and nef) was chosen as a target for triplex-forming oligonucleotides in in vitro transcription assays. Inhibition of transcription elongation was observed with triplex-forming oligonucleotide-acridine conjugates (Acr-15-TCG:5'-Acr-T4CT4G6-3' and Acr-9-TC:5'-Acr-T4CT4-3' where C is 5-methylcytosine) under conditions where the unsubstituted oligomers did not exhibit any inhibitory effect. Both SP6 bacteriophage RNA polymerase and eukaryotic RNA polymerase II were physically blocked by such a triplex barrier. The polymerase arrest is caused by the triple-helical complex involving the hydrogen-bonded oligonucleotide stabilized by the intercalated moiety and not solely by the acridine molecule specifically intercalated at the duplex-triplex junction. The stability of the triple-helical complex formed by the 15-mer containing thymines, cytosine, and guanines (15-TCG) and involving the formation of six contiguous C.GxG base triplets was strongly enhanced in the presence of a benzopyridoindole derivative (BePI), which intercalates in triplex structures. This improvement of the binding affinity led to an increased inhibition of transcription elongation. The present results demonstrate the necessity to use triplex-forming oligonucleotides with high binding affinity and a long residence time on their double-stranded target to efficiently inhibit transcription elongation. These data provide a rational basis for the optimization and the development of triplex-forming oligonucleotides as transcriptional blockers, even when they are targeted to the transcribed portion of a gene, downstream of the transcription initiation site.

Base Sequence↗
PubMed1996

Stable triple helices formed by oligonucleotide N3'-->P5' phosphoramidates inhibit transcription elongation.

Oligonucleotide analogs with N3'-->P5' phosphoramidate linkages bind to the major groove of double-helical DNA at specific oligopurine.oligopyrimidine sequences. These triple-helical complexes are much more stable than those formed by oligonucleotides with natural phosphodiester linkages. Oligonucleotide phosphoramidates containing thymine and cytosine or thymine, cytosine, and guanine bind strongly to the polypurine tract of human immunodeficiency virus proviral DNA under physiological conditions. Site-specific cleavage by the Dra I restriction enzyme at the 5' end of the polypurine sequence was inhibited by triplex formation. A eukaryotic transcription assay was used to investigate the effect of oligophosphoramidate binding to the polypurine tract sequence on transcription of the type 1 human immunodeficiency virus nef gene under the control of a cytomegalovirus promoter. An efficient arrest of RNA polymerase II was observed at the specific triplex site at submicromolar concentrations.

Amides↗
PubMed1993

Oligonucleotide clamps arrest DNA synthesis on a single-stranded DNA target.

Triple helices can be formed on single-stranded oligopurine target sequences by composite oligonucleotides consisting of two oligonucleotides covalently linked by either a hexaethylene glycol linker or an oligonucleotide sequence. The first oligomer forms Watson-Crick base pairs with the target, while the second oligomer engages in Hoogsteen base pairing, thereby acting as a molecular clamp. The triple-helical complex formed by such an oligonucleotide clamp, or "oligonucleotide-loop-oligonucleotide" (OLO), is more stable than either the corresponding trimolecular triple helix or the double helix formed upon binding of the oligopyrimidine complement to the same oligopurine target. Attaching a psoralen derivative to the 5' end of the OLO allowed us to photoinduce a covalent linkage to the target sequence. The psoralen moiety became covalently linked to all three portions of the triplex, thereby making the oligonucleotide clamp irreversible. These crosslinking reactions introduced strong stop signals during DNA replication, as shown on a plasmid containing a portion of the HIV proviral sequence of human immunodeficiency virus. A 16-mer oligopurine sequence corresponding to the "polypurine tract" of human immunodeficiency virus was chosen as a target for a psoralen-OLO conjugate. Three different stop signals for DNA polymerase were observed, corresponding to different sites of polymerase arrest on its template. Even in the absence of photoinduced crosslinking, the psoralen-OLO conjugate was able to arrest DNA replication. The formation of triple-helical structures on single-stranded targets may provide an alternative to the antisense strategy for the control of gene expression.

Base Composition↗
PubMed1992

Kinetics and thermodynamics of triple-helix formation: effects of ionic strength and mismatches.

Thermodynamic and kinetic parameters for the triplex-forming reactions between a homopurine-homopyrimidine 22-base-pair duplex (sequence of the purine strand: 5'd[AAAGGAGGAGAAGAAGAAAAAA]3') and the four 22-dN third strands (22 dN: 5'd[TTTCCTCCTCTNCTTCTTTTTT]3', where N = A, C, T, or G) were determined from thermal denaturation and renaturation UV absorbance profiles. Cooling and heating curves were not superimposable and thus allowed us to determine the rate constants of association (k(on)) and dissociation (k(off)) as a function of temperature, assuming a two-state model analogous to that developed for duplex-forming reactions. Experiments were performed in 10 mM cacodylate buffer (pH 6.8) in the presence of NaCl concentrations ranging from 20 to 300 mM. Within experimental accuracy, the main results are the following: (i) The rate constants k(on) and k(off) result in linear Arrhenius plots, consistent with the prediction of two-state association and dissociation (ii) k(on) is independent of the nature of the base N located in the center of the third strand. (iii) k(on) strongly decreases when the NaCl concentration is decreased. (iv) The activation energy, E(on), is always negative and becomes more negative when the NaCl concentration is decreased. (v) k(off) is independent of NaCl concentration but depends on the base N, with its magnitude following the order C greater than G greater than A much greater than T. (vi) The activation energy, E(off), is independent of the base N. All these results are discussed in the light of a nucleation-zipping model similar to that developed for the duplex-coil transitions [Craig, M. E., Crothers, D. M., & Doty, P. (1971) J. Mol. Biol. 62, 383-401; Pörschke, D., Eigen, M. (1971) J. Mol. Biol. 62, 361-381].

Base Composition↗