Efficient Inhibition of Transcription Elongationin vitroby Oligonucleotide Phosphoramidates Targeted to Proviral HIV DNA

Giovannangéli, Carine; Perrouault, L.; Escudé, Christophe; Gryaznov, Sergei M.; Hélène, Claude

doi:10.1006/jmbi.1996.0471

Cited by 64 publications

(30 citation statements)

References 38 publications

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“…Plasmids-The pSP-F47 plasmid was constructed by insertion of a 780-bp fragment of the human immunodeficiency virus type 1/nef gene containing the oligopyrimidine⅐oligopurine PPT target sequence between the T 7 and SP 6 promoters in the pSP73 host vector (Promega), as previously described in detail (18). This system allowed us to run bidirectional in vitro transcription assays.…”

Section: Methodsmentioning

confidence: 99%

Exploring Cellular Activity of Locked Nucleic Acid-modified Triplex-forming Oligonucleotides and Defining Its Molecular Basis

Brunet

Alberti

Perrouault

et al. 2005

Journal of Biological Chemistry

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Triplex-forming oligonucleotides (TFOs), as DNAbinding molecules that recognize specific sequences, offer unique potential for the understanding of processes occurring on DNA and associated functions. They are also powerful DNA recognition elements for the positioning of ubiquitous molecules acting on DNA, such as anticancer drugs. A prerequisite for further development of DNA code-reading molecules including TFOs is their ability to form a complex in a cellular context: their binding affinities must be comparable to those of DNA-associated proteins. To reach this goal, chemically modified TFOs must be developed. In this work, we present triplex-forming properties (kinetics and thermodynamics) and cellular activity of G-containing locked nucleic acid-modified TFOs (TFO/LNAs). In conditions simulating physiological ones, these TFO/LNAs strongly enhanced triplex stability compared with the non-modified TFO or with the pyrimidine TFO/LNA directed against the same oligopyrimidine⅐oligopurine sequence, mainly by decreasing the dissociation rate constant and conferring an entropic gain. We provide evidence of their biological activity by a triplex-based mechanism, in vitro and in a cellular context, under conditions in which the parent phosphodiester oligonucleotide did not exhibit any inhibitory effect.The design of synthetic non-protein molecules able to control DNA-associated biological functions through their interaction with a specific DNA sequence represents a very attractive approach. Among DNA code-reading molecules, triplex-forming oligonucleotides (TFOs) 1 are able to bind to the major groove of oligopyrimidine⅐oligopurine regions in doublestranded DNA. A series of results have validated triplex-based approaches at the molecular and cellular levels: triplexes have been shown to interfere with transcription (initiation and elongation), replication, repair, and recombination (1, 2). However, the intracellular efficiency of TFOs still has to be improved.One possible approach consists of increasing the stability of the non-covalent triple helices under physiological conditions to reach binding affinities comparable to those of DNA-associated regulatory proteins. To this end, a variety of chemically modified nucleic acids have been developed. Among them are locked nucleic acids (LNAs) that contain LNA nucleotide monomers, i.e. ribonucleotides with a 2Ј-O,4Ј-C-methylene linkage that effects conformational fixation of the furanose ring in a C3Ј-endo conformation (3, 4). LNA-containing oligonucleotides have been recently shown to enhance triplex stability and to alleviate in part the sequence constraints imposed by the triple helical recognition motifs (5, 6). Only a few hybridization properties of LNA-modified TFOs (TFO/LNAs) have been reported so far, and they all concern (T,C)-containing TFO/LNAs. It has been shown that fully modified TFO/LNAs failed to bind to double-stranded DNA (7, 8), likely due to conformational restraint of TFO/LNA. However, alternating DNA and LNA nucleotides in TFO sequences is appropri...

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Section: Methodsmentioning

confidence: 99%

Exploring Cellular Activity of Locked Nucleic Acid-modified Triplex-forming Oligonucleotides and Defining Its Molecular Basis

Brunet

Alberti

Perrouault

et al. 2005

Journal of Biological Chemistry

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“…To our knowledge no report has described the arrest of transcription elongation by TFOs, most probably because unmodified oligonucleotides do not form stable enough complexes with their DNA target. We previously have reported that oligomers with N3Ј-P5Ј np linkages formed more stable triplexes and that transcription elongation could be inhibited in an in vitro assay using cell extracts (10,11). The demonstration that TFOs could inhibit transcription elongation would considerably extend the range of applications of the antigene strategy in as much as both intronic and exonic sequences could be targeted in genes of interest.…”

Section: Demonstration Of Triplex Involvement In Inhibition Of Transcmentioning

confidence: 98%

“…In the present study we report triplex-induced inhibition of transcription elongation in cell cultures using oligonucleotide analogues containing N3Ј-P5Ј phosphoramidate (np) linkages that we previously have characterized as the best blockers of RNA synthesis in vitro (10,11). To unambiguously demonstrate that triplex formation is the molecular mechanism by which TFOs inhibit gene expression we have designed cellular systems that differ only by the presence or the absence of the oligopyrimidine⅐oligopurine target site, located either in a transfected plasmid or in an endogenous gene.…”

mentioning

confidence: 99%

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

Faria

Wood

Perrouault

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

139

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Triple-helix-forming oligonucleotides (TFOs) bind in the major groove of double-stranded DNA at oligopyrimidine⅐oligopurine 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 PPT͞HIV-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 PPT͞HIV-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.

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“…Sugar modifications that enhance the stability in vitro of pyrimidine motif triplexes have been characterized recently (71). In addition, oligonucleotides with novel backbones can form quite stable triplexes (72,73). Purine TFOs with base and backbone modifications that increase triplex stability have also been described (40,74).…”

Section: Discussionmentioning

confidence: 99%

Stability of DNA Triplexes on Shuttle Vector Plasmids in the Replication Pool in Mammalian Cells

Liu¹,

Majumdar²,

Klotz³

et al. 2000

Journal of Biological Chemistry

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Triple helix-forming oligonucleotides may be useful as gene-targeting reagents in vivo, for applications such as gene knockout. One important property of these complexes is their often remarkable stability, as demonstrated in solution and in cells following transfection. Although encouraging, these measurements do not necessarily report triplex stability in cellular compartments that support DNA functions such as replication and mutagenesis. We have devised a shuttle vector plasmid assay that reports the stability of triplexes on DNA that undergoes replication and mutagenesis. The assay is based on plasmids with novel variant supF tRNA genes containing embedded sequences for triplex formation and psoralen cross-linking. Triple helix-forming oligonucleotides were linked to psoralen and used to form triplexes on the plasmids. At various times after introduction into cells, the psoralen was activated by exposure to long wave ultraviolet light (UVA). After time for replication and mutagenesis, progeny plasmids were recovered and the frequency of plasmids with mutations in the supF gene determined. Site-specific mutagenesis by psoralen cross-links was dependent on precise placement of the psoralen by the triple helix-forming oligonucleotide at the time of UVA treatment. The results indicated that both pyrimidine and purine motif triplexes were much less stable on replicated DNA than on DNA in vitro or in total transfected DNA. Incubation of cells with amidoanthraquinone-based triplex stabilizing compounds enhanced the stability of the pyrimidine triplex.Triple helix-forming oligonucleotides (TFOs), 1 or third strands, have received considerable attention because of their potential for intracellular gene targeting (1-11). Many of the biochemical and biophysical properties of triplexes appear to support this application. Triplex formation following the encounter between an appropriate third strand and duplex target has long been recognized as a fundamental structural option of nucleic acids and is not dependent on enzymes or proteins (12). The most stable triplexes are formed on polypurine:polypyrimidine sequences. However, there is considerable stringency with respect to the specific sequence such that single interruptions in a polypurine run can be destabilizing (13-15). Depending on the actual sequence of the purine:pyrimidine run, triplexes can be formed by third strands composed of either purines or pyrimidines (16), which offers some flexibility in TFO design.Once formed, triplexes can be quite stable under appropriate conditions. This was demonstrated some time ago with a pyrimidine triplex with a dissociation half-life of many hours (17). Some purine TFOs form even more stable complexes, with melting temperatures equivalent to or greater than the target duplex (18), and with half-lives of days under optimal conditions (19,20). The persistence of purine motif triplexes formed on DNA fragments and then introduced into mammalian cells has also been measured. A footprinting assay to measure triplexes on transfected...

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Efficient Inhibition of Transcription Elongationin vitroby Oligonucleotide Phosphoramidates Targeted to Proviral HIV DNA

Cited by 64 publications

References 38 publications

Exploring Cellular Activity of Locked Nucleic Acid-modified Triplex-forming Oligonucleotides and Defining Its Molecular Basis

Exploring Cellular Activity of Locked Nucleic Acid-modified Triplex-forming Oligonucleotides and Defining Its Molecular Basis

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

Stability of DNA Triplexes on Shuttle Vector Plasmids in the Replication Pool in Mammalian Cells

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