Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities

Triplex-forming oligonucleotides (TFOs) are powerful tools to interfere sequence-specifically with DNA-associated biological functions. (A/T,G)-containing TFOs are more commonly used in cells than (T,C)-containing TFOs, especially C-rich sequences; indeed the low intracellular stability of the non-covalent pyrimidine triplexes make the latter less active. In this work we studied the possibility to enhance DNA binding of (T,C)-containing TFOs, aiming to reach cellular activities; to this end, we used locked nucleic acid-modified TFOs (TFO/LNAs) in association with 5′-conjugation of an intercalating agent, an acridine derivative. In vitro a stable triplex was formed with the TFO-acridine conjugate: by SPR measurements at 37°C and neutral pH, the dissociation equilibrium constant was found in the nanomolar range and the triplex half-life ∼10 h (50-fold longer compared with the unconjugated TFO/LNA). Moreover to further understand DNA binding of (T,C)-containing TFO/LNAs, hybridization studies were performed at different pH values: triplex stabilization associated with pH decrease was mainly due to a slower dissociation process. Finally, biological activity of pyrimidine TFO/LNAs was evaluated in a cellular context: it occurred at concentrations ∼0.1 μM for acridine-conjugated TFO/LNA (or ∼2 μM for the unconjugated TFO/LNA) whereas the corresponding phosphodiester TFO was inactive, and it was demonstrated to be triplex-mediated

Sequence-specific DNA cleavage mediated by bipyridine polyamide conjugates

The design of molecules that damage a selected DNA sequence provides a formidable opportunity for basic and applied biology. For example, such molecules offer new prospects for controlled manipulation of the genome. The conjugation of DNA-code reading molecules such as polyamides to reagents that induce DNA damages provides an approach to reach this goal. In this work, we showed that a bipyridine conjugate of polyamides was able to induce sequence-specific DNA breaks in cells. We synthesized compounds based on two polyamide parts linked to bipyridine at different positions. Bipyridine conjugates of polyamides were found to have a high affinity for the DNA target and one of them produced a specific and high-yield cleavage in vitro and in cultured cells. The bipyridine conjugate studied here, also presents cell penetrating properties since it is active when directly added to cell culture medium. Harnessing DNA damaging molecules such as bipyridine to predetermined genomic sites, as achieved here, provides an attractive strategy for targeted genome modification and DNA repair studies

Flavin Conjugates for Delivery of Peptide Nucleic Acids

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Sequences of the oligopyrimidine•oligopurine DNA targets () and of the oligonucleotides () used in this study

<p><b>Copyright information:</b></p><p>Taken from "Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities"</p><p>Nucleic Acids Research 2005;33(13):4223-4234.</p><p>Published online 27 Jul 2005</p><p>PMCID:PMC1181241.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> (A) The wild-type PPT target sequence (boxed), Dra I recognition site (underlined) are shown, as well as the mutated PPT duplex presenting two mutations (in bold). (B) (T,C)-containing TFOs directed against the wild-type PPT duplex and control sequences. Small letters indicate LNA nucleotides and capitals DNA nucleotides. Cytosines in italic ( and ) are methylated at position 5; all cytosines (LNA and DNA) are 5-methylated. The abbreviated names are indicated near the sequences. (po) stands for phosphodiester. Last four TFOs are either acridine (Acr) or psoralen (Pso) 5′-conjugated TFO/LNAs

Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9 Nucleases

Genome editing has now been reported in many systems using TALEN and CRISPR-Cas9 nucleases. Precise mutations can be introduced during homology-directed repair with donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for gene knockout and optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified oligonucleotides strongly enhances genome editing efficiency of single-stranded oligonucleotide donors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. Finally, when detected, imprecise knockin events exhibit indels that are asymmetrically positioned, consistent with genome editing taking place by two steps of single-strand annealing