New junction models for alternate-strand triple-helix formation

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Oligonucleotide-directed triple-helix (triplex) formation is a strategy to interfere with gene expression.
• Current methods are limited to targeting long oligopyrimidine*oligopurine sequences.
• Previous attempts to target shorter, alternating sequences involved covalently linking two triplex-forming oligonucleotides.

Purpose of the Study:

• To rationally optimize alternate-strand triplex formation on DNA duplexes.
• To target specific DNA sequences with alternating purines on opposite strands.
• To investigate binding strategies for 5'-GpT-3'/3'-CpA-5' and 5'-TpG-3'/3'-ApC-5' steps.

Main Methods:

• Utilizing a combination of (G,T)- and (G,A)-containing oligonucleotides.
• Designing oligonucleotides to bind to oligopurine strands in opposite orientations.
• Employing nucleotide deletion and linker addition strategies.

Main Results:

• A single nucleotide deletion in the reverse Hoogsteen region optimized binding at the 5'GpT-3'/3'-CpA-5' step.
• Adding two cytosines as a linker effectively crossed the 5'-TpG-3'/3'-ApC-5' step.
• Experimental data and energy minimization suggest the linker cytosines form novel base quadruplets.

Conclusions:

• Established a rational design basis for oligonucleotides targeting alternating purine sequences.
• Demonstrated effective triplex formation at specific DNA duplex junctions (5'-GpT-3'/3'-CpA-5' and 5'-TpG-3'/3'-ApC-5').
• Provided insights into novel base quadruplet formation in triplex structures.