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

While sequence-selective dsDNA targeting by triplex forming oligonucleotides has been studied extensively, only very little is known about the properties of PNA-dsDNA triplexes-mainly due to the competing invasion process. Here we show that when appropriately modified using pseudoisocytosine substitution, in combination with (oligo)lysine or 9-aminoacridine conjugation, homopyrimidine PNA oligomers bind complementary dsDNA targets via triplex formation with (sub)nanomolar affinities (at pH 7.2, 150 mM Na+). Binding affinity can be modulated more than 1000-fold by changes in pH, PNA oligomer length, PNA net charge and/or by substitution of pseudoisocytosine for cytosine, and conjugation of the DNA intercalator 9-aminoacridine. Furthermore, 9-aminoacridine conjugation also strongly enhanced triplex invasion. Specificity for the fully matched target versus one containing single centrally located mismatches was more than 150-fold. Together the data support the use of homopyrimidine PNAs as efficient and sequence selective tools in triplex targeting strategies under physiological relevant conditions.