Biochemical evidence that a D-loop is part of a four-stranded PNA-DNA bundle. Nickel-mediated cleavage of duplex DNA by a Gly-Gly-His Bis-PNA

A peptide nucleic acid (PNA) with improved strand-displacement capability and a site-specific DNA cleavage function is a novel reagent for probing the structure of PNA-DNA complexes in solution. By linking two PNAs in tandem with an aliphatic linker, the bis-PNA forms a bis-PNA-DNA triple-stranded complex having a higher stability to thermal denaturation than conventional monomeric PNAs. When a Gly-Gly-His tripeptide is placed on either the Watson-Crick or Hoogsteen bis-PNA strand, nickel-mediated cleavage is detected at specific sites on the displaced and hybridized DNA strands. Because the displaced strand is cleaved when GGH is placed on either PNA strand, the D-loop must be close to the backbone of the bis-PNA-DNA tripler. Furthermore, the pattern of cleavage on the displaced strand suggests the nickel-tripeptide complex lies in a groove formed by the displaced DNA strand and both PNA strands. These observations suggest that the D-loop is a part of a four-stranded bis-PNA-DNA(2) bundle.