Recognition of Non-standard Base Pairs by Triplex-Forming Oligonucleotides Containing an Expanded Genetic Alphabet

Area of Science:

• Synthetic Biology
• Molecular Medicine
• Biochemistry

Background:

• Sequence-specific DNA recognition is crucial for molecular medicine and synthetic biology.
• Triplex-forming oligonucleotides (TFOs) offer programmable DNA recognition but are limited by natural nucleobases and require acidic conditions.
• Expanding the DNA recognition repertoire is essential for broader applications.

Purpose of the Study:

• To expand the targeting capabilities of TFOs beyond natural nucleobases.
• To identify new DNA recognition triplets for use at neutral pH.
• To demonstrate the utility of an expanded genetic alphabet for DNA sensing and targeting.

Main Methods:

• Systematic evaluation of 120 base triplet combinations from an artificially expanded genetic information system (AEGIS).
• Chemical and enzymatic synthesis of TFOs.
• Detection of DNA lesions and AEGIS base pairs in assembled DNA constructs.

Main Results:

• Identification of at least 12 new modular triplets for DNA recognition.
• Achieved nanomolar affinity for targeting duplex DNA with standard, damaged, or synthetic base pairs at neutral pH.
• Demonstrated detection of oxidative lesions and AEGIS base pairs using AEGIS-modified TFOs.

Conclusions:

• The expanded AEGIS recognition code significantly broadens the scope of TFO-mediated DNA targeting.
• This generalized framework supports precision gene targeting, molecular sensing, and nucleic acid nanotechnology applications.
• The development enables robust and versatile DNA recognition under physiological conditions.