Preparation and Application of Triple Helix Forming Oligonucleotides and Single Strand Oligonucleotide Donors for Gene Correction

Abstract

Strategies for site-specific modulation of genomic sequences in mammalian cells require two components. One must be capable of recognizing and activating a specific target sequence in vivo, driving that site into an exploitable repair pathway. Information is transferred to the site via participation in the pathway by the second component, a donor nucleic acid, resulting in a permanent change in the target sequence. We have developed biologically active triple helix forming oligonucleotides (TFOs) as site-specific gene targeting reagents. These TFOs, linked to DNA reactive compounds (such as a cross-linking agent), activate pathways that can engage informational donors. We have used the combination of a psoralen-TFO and single strand oligonucleotide donors to generate novel cell lines with directed sequence changes at the target site. Here we describe the synthesis and purification of bioactive psoralen-linked TFOs, their co-introduction into mammalian cells with donor nucleic acids, and the identification of cells with sequence conversion of the target site. We have emphasized details in the synthesis and purification of the oligonucleotides that are essential for preparation of reagents with optimal activity.

The FANC/BRCA Pathway Releases Replication Blockades by Eliminating DNA Interstrand Cross-Links

“…Based on DNA single fiber analysis [147] conducted by spreading and/or combing of DNA extracted from human cells following the induction of ICLs by the photoactivation of biotin-labeled psoralens [183][184][185], the groups of Seidman and Wang suggested that the major pathway involved in replication-associated ICL repair depends on a FANCM-mediated ICL traverse [146]. A single-molecule analysis allowed them to observe that, in the large majority of cases, the fork encountering an ICL was able to "traverse" it without repairing the lesion (Figure 3C).…”

Versatile and efficient chromatin pull-down methodology based on DNA triple helix formation

“…Nearly 30 years later, formation of DNA triplex was also reported 13 . Since then, the properties of triplexes have been intensively studied because they potentially offer attractive applications for engineering and therapeutic purposes 14 – 16 . The key feature of triplexes is that a third strand (referred to as Triplex Forming Oligonucleotide (TFO)) consisting of either a pyrimidine-rich or a purine-rich sequence, forms Hoogsteen (or reverse Hoogsteen) base pairs with a complementary purine-rich strand in the major groove of double-stranded DNA (dsDNA) without disrupting its canonical Watson-Crick base pairs.…”