Triple helix formation by (G,A)-containing oligonucleotides: asymmetric sequence effect

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Triple helix formation is crucial for DNA recognition and manipulation.
• Purine-motif (G,A)-motif triple helices are important structures in nucleic acid research.
• Triplex-forming oligonucleotides (TFOs) can bind to specific DNA sequences.

Purpose of the Study:

• To investigate sequence effects on the stability of purine-motif triple helices.
• To understand how TFO self-association influences triple helix formation.
• To determine the impact of TFO length and terminal sequences on binding affinity.

Main Methods:

• Synthesis and characterization of TFOs with specific core sequences (5'(GGA)43' and 5'(AGG)43').
• Assessment and minimization of TFO self-association.
• Systematic variation of TFO lengths and terminal base sequences.
• Evaluation of TFO binding affinity to target double-stranded DNA (dsDNA).

Main Results:

• Triple helix stability varied significantly (up to 6-fold) between reverse strand orientations.
• Extending the 5'(GGA)43' core at the 3'-end with a G enhanced binding affinity compared to extensions with A.
• Extending the 5'(AGG)43' core at the 5'-end with additional bases increased binding affinity.
• Terminal base triplets critically influenced triplex stability, particularly at the 3'-end nucleation site.

Conclusions:

• Sequence and strand orientation are critical determinants of (G,A)-motif triple helix stability.
• TFO length and terminal sequence modifications can be used to optimize binding affinity.
• The 3'-terminal base triplet plays a key role in the nucleation and overall stability of triple helices.