Ligand-Mediated Transcription Elongation Control Using Triplex-Based Padlock Oligonucleotides

Abstract

Triplex-forming oligonucleotides (TFOs) provide useful tools for the artificial regulation of gene expression at the transcriptional level. They can become topologically linked to their DNA target upon circularization, thereby forming very stable triple helical structures. These "padlock oligonucleotides" are able to interfere with transcription elongation when their target site is located in the transcribed region of a gene. In vitro transcription experiments showed that a bacterial RNA polymerase was stopped at the site of triple-helix formation, whereas expression of a reporter gene was inhibited in live cells. In both cases, the padlock oligonucleotide was more efficient at inhibiting transcription elongation than a linear TFO, and the inhibition was observed only in the presence of a triplex stabilizing agent. These results provide new insights into the ligand-modulated locking of padlock oligonucleotides around their DNA target.

Stem–loop oligonucleotides as tools for labelling double‐stranded DNA

“…2). The first type contains G and T in the loop of the TFO and can form a triple helix in the presence of the triplex‐stabilizing agent 6‐[3‐(dimethylamino)propyl]amino‐11‐methoxy‐benzo[f]quino‐[3,4‐b]quinoxaline (BQQ), as already described in our previous work [10–14]. The second one can form a triple helix at acidic pH.…”

“…We have shown that the stability of the triple helix made by the topologically linked TFO, also called padlock oligonucleotide, was enhanced compared to that formed with a linear TFO [10]. For example, we showed that such a padlock oligonucleotide strongly inhibits DNA digestion by a restriction endonuclease, and that the complex is strong enough to inhibit the elongation of transcription by an RNA polymerase [11]. The triple helix used in these studies involved a third strand containing G and T, binding of which was stabilized by the use of a triplex specific intercalator.…”

DNA Major Groove Binders: Triple Helix-Forming Oligonucleotides, Triple Helix-Specific DNA Ligands and Cleaving Agents

“…9). In the absence of BQQ, the circular oligonucleotide was free to move from its binding site, whereas addition of BQQ locked the oligonucleotide around its target, resulting in inhibition of cleavage by a restriction enzyme [143] and blockage of transcription elongation by an RNA polymerase [144]. This approach could lead to new strategies aimed at regulating gene expression by small molecules.…”

A New Supramolecular Structure Made of Two Different Plasmids Linked by a Circular Oligonucleotide

“…We chose another plasmid, pRLCMV (4079 bp), on which another oligopurine sequence, 5'-GAAGAAGGAGAAAAAA-3', provides a target for TFO2. [12] The sequences of the two 5'-phosphorylated oligonucleotides are shown in Figure 1. The design of the TFOs resembles the one described in some of our previous work.…”

“…[10] The circularized oligonucleotide might remain locked on its target sequence or slide on the DNA double-helix depending on whether conditions favor triple-helix formation or not. [11,12] The so-called padlock oligonucleotides for double-stranded DNA might have several applications, including plasmid targeting [13] and sequence-specific labeling of DNA for observation by electron, atomic force, or optical microscopy. [14,15] Here, we report the preparation and characterization of a novel supramolecular plasmid DNA construct containing two different plasmid molecules that are linked by a circular oligonucleotide.…”