DNA nanostructures are ordered oligonucleotide arrangements that have applications for DNA computers, crystallography, diagnostics and material sciences. Peptide nucleic acid (PNA) is a DNA/RNA mimic that offers many advantages for hybridization, but its potential for application in the field of DNA nanotechnology has yet to be thoroughly examined. We report the synthesis and characterization of tethered PNA molecules (bisPNAs) designed to assemble two individual DNA molecules through Watson-Crick base pairing. The spacer regions linking the PNAs were varied in length and contained amino acids with different electrostatic properties. We observed that bisPNAs effectively assembled oligonucleotides that were either the exact length of the PNA or that contained overhanging regions that projected outwards. In contrast, DNA assembly was much less efficient if the oligonucleotides contained overhanging regions that projected inwards. Surprisingly, the length of the spacer region between the PNA sequences did not greatly affect the efficiency of DNA assembly. Reasons for inefficient assembly of inward projecting DNA oligonucleotides include non-sequence-specific intramolecular interactions between the overhanging region of the bisPNA and steric conflicts that complicate simultaneous binding of two inward projecting strands. These results suggest that bisPNA molecules can be used for self-assembling DNA nanostructures provided that the arrangement of the hybridizing DNA oligonucleotides does not interfere with simultaneous hybridization to the bisPNA molecule.
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