High-affinity triplex targeting of double stranded DNA using chemically modified peptide nucleic acid oligomers

Area of Science:

• Biochemistry
• Molecular Biology
• Oligonucleotide Chemistry

Background:

• Triplex forming oligonucleotides (TFOs) are known for dsDNA targeting.
• Properties of PNA-dsDNA triplexes are poorly understood due to competing invasion.
• Homopyrimidine PNA oligomers are investigated for dsDNA binding.

Purpose of the Study:

• To investigate the properties of PNA-dsDNA triplexes.
• To explore modifications enhancing PNA-dsDNA triplex formation and stability.
• To assess sequence selectivity and binding affinities under physiological conditions.

Main Methods:

• Synthesis of modified homopyrimidine PNA oligomers.
• Pseudoisocytosine substitution and conjugation with (oligo)lysine or 9-aminoacridine.
• Affinity and specificity measurements of PNA-dsDNA triplex formation.
• pH, ionic strength, and PNA modification effects on binding.

Main Results:

• Modified PNAs achieve (sub)nanomolar binding affinities for dsDNA targets via triplex formation.
• Binding affinity is highly tunable (>1000-fold) by pH, PNA length, charge, and modifications.
• Pseudoisocytosine substitution and 9-aminoacridine conjugation enhance affinity and invasion.
• High specificity (>150-fold) observed for matched targets over mismatched ones.

Conclusions:

• Homopyrimidine PNAs, when appropriately modified, are effective tools for sequence-selective dsDNA triplex targeting.
• These PNA-dsDNA triplexes exhibit high affinity and specificity under physiological conditions.
• The findings support the use of modified PNAs in triplex-based targeting strategies.