Targeting linear duplex DNA with mixed-base peptide nucleic acid oligomers facilitated by bisPNA openers.

PD-loop complexes formed by bisPNA openers and a mixed-base (mb) oligonucleotide on double-stranded (ds) DNA enable various diagnostic and biotechnological manipulations with linear DNA duplexes [1–5]. So far, the PD-loop formation requires the use of two homopyrimidine bisPNA oligomers to open the DNA duplex for binding an oligonucleotide [6]. This condition imposes certain sequence limitations on the PD-loop-forming sites. Besides, substantial overlap between the bisPNA and oligonucleotide sequences is an unavoidable feature of PD-loops. Indeed, only those oligonucleotides that are longer than 10 nt can form suYciently stable hybridization complexes within the PD-loops, whereas for their eYcient formation the peptide nucleic acid (PNA) openers cannot be separated by more than 10 bp [6].2 Such sequence overlap necessitates the sequential dsDNA targeting Wrst by PNA openers with subsequent removal of unbound bisPNAs followed by targeting by an oligonucleotide. Otherwise, PNA openers would obstruct the oligonucleotide binding to dsDNA if targeted simultaneously and/or without removal of unbound PNA oligomers. These two major requirements on the PD-loop formation limit their applications. Here we demonstrate that PPloops, i.e., complexes similar to PD-loops but formed by