Triple helix formation inhibits transcription elongation in vitro.

Young, Sharon; Krawczyk, Steven H.; Matteucci, Mark D.; Toole, John J.

doi:10.1073/pnas.88.22.10023

Cited by 186 publications

(97 citation statements)

References 19 publications

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“…Resolution of the D-loop is then completed when the template and nontemplate strands reanneal as usual at the distal end of the transcription bubble. RNA polymerase can also transcribe through most triple-stranded complexes (7,29,39), presumably dissociating the oligonucleotide in the process. In D-loops where the oligonucleotide is hybridized to the nontemplate strand of the gene, transcription can proceed without release of the oligonucleotide.…”

Section: Discussionmentioning

confidence: 99%

Strand Bias in Targeted Gene Repair Is Influenced by Transcriptional Activity

Liu

Rice

Drury

et al. 2002

Molecular and Cellular Biology

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Modified single-stranded DNA oligonucleotides can direct nucleotide exchange in Saccharomyces cerevisiae. Point and frameshift mutations are corrected in a reaction catalyzed by cellular enzymes involved in various DNA repair processes. The present model centers on the annealing of the vector to one strand of the helix, followed by the correction of the designated base. The choice of which strand to target is a reaction parameter that can be controlled, so here we investigate the properties of strand bias in targeted gene repair. An in vivo system has been established in which a plasmid containing an actively transcribed, but mutated, hygromycinenhanced green fluorescent protein fusion gene is targeted for repair and upon conversion will confer hygromycin resistance on the cell. Overall transcriptional activity has a positive influence on the reaction, elevating the frequency. If the targeting vector is synthesized so that it directs nucleotide repair on the nontranscribed strand, the level of gene repair is higher than if the template strand is targeted. We provide data showing that the targeting vector can be displaced from the template strand by an active T7 phage RNA polymerase. The strand bias is not influenced by which strand serves as the leading or lagging strand during DNA synthesis. These results may provide an explanation for the enhancement of gene repair observed when the nontemplate strand is targeted.The use of oligonucleotides to direct single-base changes in DNA has become an important strategy for understanding gene function. While processes for disabling targeted genes and creating knockout strains have provided an enormous amount of information, it is now becoming evident that subtle changes in the genomes of many organisms constitute a centerpiece of genetic variability. Specifically, as information regarding the human genome accumulates, the importance of single-nucleotide polymorphisms is now clear. These simple alterations, often causing no visible phenotype variation, may hold the key to the spectrum of responses to many therapeutic agents. In recognition of the importance of single-nucleotide polymorphisms, several technologies have emerged that aim to create animal and plant models that will recapitulate the nucleotide alterations without disrupting the integrity of the whole gene. Many of these technologies employ synthetic oligonucleotides as vectors for directing the introduction of the specific changes.Our laboratory has developed a series of these oligonucleotide vectors that can create single-base changes in chromosomal and episomal targets. One of these vectors is the chimeric RNA/DNA oligonucleotide (chimera), which consists of cRNA and DNA residues folded into a double hairpin conformation. Chimeras have been shown to direct base replacement or base insertion in yeast (21, 27), mammalian cells in culture (1, 6, 15, 38), animal models (2, 14, 16, 26, 31), and plants (3,28,40). While the mechanism of targeted gene repair is not fully elucidated, a number of important steps have...

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Section: Discussionmentioning

confidence: 99%

Strand Bias in Targeted Gene Repair Is Influenced by Transcriptional Activity

Liu

Rice

Drury

et al. 2002

Molecular and Cellular Biology

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“…Formation ofpsoralen photoproducts (15)(16)(17), as well as a stable triplex on a duplex target, has been shown to inhibit transcription (18,19). On a single-stranded nucleic acid, triplex formation might be expected to be more efficient in arresting biological processes than formation of a double helix with an antisense oligonucleotide.…”

Section: Resultsmentioning

confidence: 99%

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

Giovannangéli

Thuong

Hélène

1993

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

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Triple helices can be formed on singlestranded 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 "oligonucleotide4oop-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

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“…The specificity of 1D in recognizing its target is supported by the absence of an antisense effect and by the lack of effect on MDR1 mRNA levels in cells treated with an ODN with the same sequence but opposite orientation, unable to form a triple helix in vitro. Although we were unable to detect MDR1 truncated transcripts, since the probe recognizes a part of the gene downstream of the target exon 3 from 1178 to 2561 bp [25], we suspect that the triple helix can physically block the movement of RNA polymerase on the template DNA [8,41].…”

Section: Discussionmentioning

confidence: 99%

“…The potential selective interaction within megabase DNA has suggested the possibility of exogenously modulating gene expression by intermolecular triplex formation [5,6]. Many in vitro experiments have demonstrated that triple helix-forming oligodeoxyribonucleotides (ODNs) can selectively inhibit gene transcription, either by preventing transcription factor binding to promoters [6,7] or by blocking transcription elongation [8,9]. This has led to consideration of the triple helix 'anti-gene' strategy as a new therapeutic approach to selectively suppress unwanted gene expression in vivo.…”

Section: Introductionmentioning

confidence: 99%

Effect of unmodified triple helix‐forming oligodeoxyribonucleotide targeted to human multidrug‐resistance gene mdr1 in MDR cancer cells

et al. 1994

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The human mdrl gene encodes a transmembrane glycoprotein the over-expression of which is associated with development of multidrug resistance in human tumor cells. A negative modulation of human mdrt has been attempted via a 27-met unmodified triple helix-forming oligonucleotide, named 1 D, targeted to a homopurine sequence in the coding region of the gene. By administering 10/zM of 1D we could find a significant reduction in MDR1 mRNA levels in the human drug-resistant cell line CEM-VLBI00. This effect appears to be specific and due to a transient block of RNA polymerase mediated by triple helix formation.

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Triple helix formation inhibits transcription elongation in vitro.

Cited by 186 publications

References 19 publications

Strand Bias in Targeted Gene Repair Is Influenced by Transcriptional Activity

Strand Bias in Targeted Gene Repair Is Influenced by Transcriptional Activity

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

Effect of unmodified triple helix‐forming oligodeoxyribonucleotide targeted to human multidrug‐resistance gene mdr1 in MDR cancer cells

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