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

Area of Science:

• Genomics
• Molecular Biology
• Gene Targeting

Background:

• Triplex-forming oligonucleotides (TFOs) offer a method for site-specific DNA recognition and genome manipulation.
• The limited number of known TFO target sequences (TTS) has restricted the application of TFO-based gene targeting.
• Identifying high-affinity TTS is crucial for successful gene targeting strategies.

Purpose of the Study:

• To develop and apply an algorithm for identifying high-affinity TFO target sequences (TTS) across the complete human and mouse genomes.
• To assess the prevalence and distribution of potential TTS in gene regulatory and coding regions.
• To validate the identified TTS by examining their affinity for TFO binding in selected genes relevant to cell signaling, proliferation, and carcinogenesis.

Main Methods:

• Genome-wide computational search for high-affinity TTS using a specialized algorithm.
• Analysis of TTS distribution within promoter, transcribed, and intergenic regions.
• In vitro binding affinity assays for TFOs targeting selected genes.

Main Results:

• Identification of 1.9 million potential high-affinity TTS in both human and mouse genomes.
• Discovery that over 95% of known genes in both species contain at least one potential high-affinity TTS.
• Demonstration that a significant majority of genes possess unique TTS, enabling specific targeting.
• Confirmation of high-affinity TFO binding sites in selected genes critical for cell signaling and cancer.

Conclusions:

• The vast number of identified TTS suggests that the majority of human and mouse genes can be targeted using TFOs.
• The prevalence of TTS in promoter regions indicates their potential role in gene regulation.
• This study provides a foundation for advancing TFO-based gene targeting for therapeutic applications, particularly in cancer and cell proliferation disorders.