High‐affinity triplex‐forming oligonucleotide target sequences in mammalian genomes

Abstract

Site-specific recognition of duplex DNA by triplex-forming oligonucleotides (TFOs) provides a promising approach to manipulate mammalian genomes. A prerequisite for successful gene targeting using this approach is that the targeted gene must contain specific, high-affinity TFO target sequences (TTS). To date, TTS have been identified and characterized in only approximately 37 human or rodent genes, limiting the application of triplex-directed gene targeting. We searched the complete human and mouse genomes using an algorithm designed to identify high-affinity TTS. The resulting data set contains 1.9 million potential TTS for each species. We found that 97.8% of known human and 95.2% of known mouse genes have at least one potential high-affinity TTS in the promoter and/or transcribed gene regions. Importantly, 86.5% of known human and 83% of the known mouse genes have at least one TTS that is unique to that gene. Thus, it is possible to target the majority of human and mouse genes with specific TFOs. We found substantially more potential TTS in the promoter sequences than in the transcribed gene sequences or intergenic sequences in both genomes. We selected 12 mouse genes and 2 human genes critical for cell signaling, proliferation, and/or carcinogenesis, identified potential TTS in each, and determined TFO binding affinities to these sites in vitro. We identified at least one high-affinity, specific TFO binding site within each of these genes. Using this information, many genes involved in mammalian cell proliferation and carcinogenesis can now be targeted.

DNA triple helices: Biological consequences and therapeutic potential

“…Since that time, many groups have published reports demonstrating the ability of TFOs to inhibit the expression of a number of genes in a variety of systems. Mammalian genes that have been targeted by TFOs are summarized by Wu et al (2007) [27]. However, obstacles to this approach have limited the potential of triplex technology as a consistent and reproducible method of transcriptional regulation.…”

Triplexator: Detecting nucleic acid triple helices in genomic and transcriptomic data

“…To ensure that these targets can form stable triple helices, we require a minimum guanine rate of 50% in the purine tract of the target (Mills et al 2002;Vekhoff et al 2008). In contrast to a previous study (Wu et al 2007), however, we refrain from filtering out lowcomplexity regions since it could remove potential targets. Unique TTSs are detected using Triplexator's duplicate filter, which discards TTSs that occur more than once in the genome (either as copy or subsequence of another TTS).…”

Purine– and pyrimidine–triple-helix-forming oligonucleotides recognize qualitatively different target sites at the ribosomal DNA locus

“…Putative triplex targeting sites are located in regulatory regions, mainly promoters, carrying a potential role in gene expression regulation (Goñi et al 2004(Goñi et al , 2006Wu et al 2007;Buske et al 2012). Accordingly, it has been shown that lncRNAs are targeted to these sites, recruiting chromatin modifiers and thereby regulating gene expression (Mondal et al 2015;O'Leary et al 2015;Postepska-Igielska et al 2015).…”

“…These approaches have revealed that mammalian genomes harbor numerous TTS, which are enriched at gene promoters and regulatory elements (Goñi et al 2004(Goñi et al , 2006Wu et al 2007;Buske et al 2012). In humans and mice, there is on average one specific TTS located ∼100-200 bp upstream of transcription start sites at every 1.3 kb within the genome (Goñi et al 2004;Wu et al 2007;Buske et al 2012).…”