An Oligo-Library-Based Approach for Mapping DNA-DNA Triplex Interactions In Vitro

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• DNA triplexes are crucial for gene regulation and therapeutic applications.
• Understanding the sequence and environmental factors governing triplex formation is essential.

Purpose of the Study:

• To develop and validate a deep-sequencing method (Triplex-seq) for systematically mapping DNA triplex interactions.
• To identify sequence motifs and conditions that dictate triplex formation and stability.

Main Methods:

• Utilized a large library of single-stranded DNA triplex-forming oligos (TFOs) against various double-stranded DNA triplex target sites (TTSs).
• Employed deep sequencing to analyze millions of TFO variants under different pH conditions (pH 5 and pH 7).
• Applied information analysis to identify minimal functional TFO sequences (triplex-forming motifs, TFMs).

Main Results:

• Identified G-rich triplexes forming at both pH 5 and pH 7, with higher stability at pH 5.
• Discovered a subset of TFOs forming triplexes exclusively at pH 5.
• Validated TFMs, demonstrating their role in G-rich TTS binding and the impact of single mutations on binding affinity.

Conclusions:

• Deep sequencing significantly enhances the understanding of DNA triplex binding rules.
• The findings aid in refining the DNA triplex code for potential applications in synthetic biology and therapeutics.