Methods for assessing DNA hybridization of peptide nucleic acid–titanium dioxide nanoconjugates

We describe the synthesis of peptide nucleic acid (PNA)–titanium dioxide (TiO2) nanoconjugates and several novel methods developed to investigate the DNA hybridization behaviors of these constructs. PNAs are synthetic DNA analogs resistant to degradation by cellular enzymes that hybridize to single-stranded DNA (ssDNA) with higher affinity than DNA oligonucleotides, invade double-stranded DNA (dsDNA), and form different PNA/DNA complexes. Previously, we developed a DNA–TiO2 nanoconjugate capable of hybridizing to target DNA intracellularly in a sequence-specific manner with the ability to cleave DNA when excited by electromagnetic radiation but susceptible to degradation that may lower its intracellular targeting efficiency and retention time. PNA–TiO2 nanoconjugates described in the current article hybridize to target ssDNA, oligonucleotide dsDNA, and supercoiled plasmid DNA under physiological-like ionic and temperature conditions, enabling rapid, inexpensive, sequence-specific concentration of nucleic acids in vitro. When modified by the addition of imaging agents or peptides, hybridization capabilities of PNA–TiO2 nanoconjugates are enhanced, providing essential benefits for numerous in vitro and in vivo applications. The series of experiments shown here could not be done with either TiO2–DNA nanoconjugates or PNAs alone, and the novel methods developed will benefit studies of numerous other nanoconjugate systems.