A triple helix is formed upon binding of an oligodeoxynucleotide to the major groove of duplex DNA. A benzo[e]pyridoindole derivative (BePI) strongly stabilized this structure and showed preferential binding to a triplex rather than to a duplex. Energy transfer experiments suggest that BePI intercalates within the triple helix. Sequence-specific inhibition of transcription initiation of a specific gene by Escherichia coli RNA polymerase by a triplex-forming oligodeoxynucleotide is strongly enhanced when the triplex is stabilized by BePI. Upon irradiation with ultraviolet light, BePI induces covalent modifications of the target within the triple helix structure.
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 ofthe triple helix. Substitution of5-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 >310 nm.Artificial control of gene expression at the transcriptional level is an essential strategy toward development of a genetic therapy. With this goal in mind, extensive investigations have been performed using anticancer drugs able to bind DNA, such as intercalators or minor groove ligands (1)(2)(3). A crucial question with these chemical reagents concerns the stability of their complexes (i.e., residence time) with the nucleic acid target (4); this parameter determines the efficiency of inhibition of biological processes such as transcription or replication that involve proteins moving along nucleic acids. Another major problem is the sequence specificity of a particular drug with respect to a nucleic acid target, intercalators and minor groove binders having a limited sequence specificity.For these reasons, oligonucleotides directed to a precise DNA sequence provide an interesting approach to artificially control the transcriptional process. Homopyrimidine oligonucleotides bind to complementary homopyrimidine-homopurine stretches by triple helix formation (5-9). We have studied the ability of an oligonucleotide-DNA complex to inhibit transcription by arresting Escherichia coli RNA polymerase (RNAP) in vitro. We have developed a transcription and footprinting assay on a DNA fragment carrying the bla promoter of E. coli, which enabled oligonucleotide-DNA complex formation under conditions in which the DNA was actively transcribed.Here, we present the results obtained with a 13-mer oligonucleotide directed to the E. coli P-lactamase gene under the control of the bla promoter. This promoter was used in our experiments because it contains a homopurine-homopyrimidine sequence located just downstream of the promoter site from +22 to +34 relative to the transcriptional ...
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