Peptide nucleic acid-assisted topological labeling of duplex dna

Peptide nucleic acids (PNAs) are a family of synthetic polyamide mimics of nucleic acids that offer a variety of applications. Pyrimidine bis-PNAs can be used for rational design of novel interlocked DNA nanostructures, earring labels, representing locked pseudorotaxanes or locked catenanes. These structures are created through DNA ligase-mediated catenation of duplex DNA with a circularized oligonucleotide tag at a designated DNA site. The assembly is performed via formation of the PD-loop consisting of a pair of bis-PNA openers and the probe oligonucleotide. The openers locally expose one of the two strands of duplex DNA for hybridizing the probe, whose termini are complementary to the displaced DNA strand. After hybridization, they are in juxtaposition and can subsequently be linked by DNA ligase. As a result, a true topological link forms at a precise position on the DNA double helix yielding locked, earring-like label. DNA topological labeling can be done both in solution and, for longer templates, within the agarose gel plug. Accordingly, highly localized DNA detection with rolling circle amplification of hybridization signal and effective micromanipulations with DNA duplexes become possible through precise spatial positioning of various ligands on the DNA scaffold.