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.

Triplex technology offers a useful approach for site-specific modification of gene structure and function both in vitro and in vivo. Triplex-forming oligonucleotides (TFOs) bind to their target sites in duplex DNA, thereby forming triple-helical DNA structures via Hoogsteen hydrogen bonding. TFO binding has been demonstrated to site-specifically inhibit gene expression, enhance homologous recombination, induce mutation, inhibit protein binding, and direct DNA damage, thus providing a tool for gene-specific manipulation of DNA. We have developed a flexible web-based search engine to find and annotate TFO target sequences within the human and mouse genomes. Descriptive information about each site, including sequence context and gene region (intron, exon, or promoter), is provided. The engine assists the user in finding highly specific TFO target sequences by eliminating or flagging known repeat sequences and flagging overlapping genes. A convenient way to check for the uniqueness of a potential TFO binding site is provided via NCBI BLAST. The search engine may be accessed at spi.mdanderson.org/tfo.

Current techniques for analysis of gene expression either monitor one gene at a time, for example northern hybridization or RT-PCR methods, or are designed for the simultaneous analysis of thousands of genes, for example microarray hybridization or serial analysis of gene expression. To provide a flexible, intermediate scale alternative, a PCR-based method for the rapid analysis of gene expression has been developed which allows expression changes to be determined in either a directed search of known genes, or an undirected survey of unknown genes. A single set of reagents and reaction conditions allows analyses of most genes in any eukaryote. The method is useful for assaying on the order of tens to hundreds of genes in multiple samples. Control experiments indicate reliable detection of changes in gene expression 2-fold and greater, and sensitivity of detection better than 1 in 10 000. Analyses of over 400 genes in a mouse system transgenic for the E2F1 gene have identified several new downstream targets of E2F1, including Brca1 and Cdk7, in addition to several unidentified genes that are upregulated in the transgenic mice. Changes in expression of several genes related to apoptosis suggest a possible potentiation of apoptotic pathways in the transgenic keratinocytes.