Peptide Nucleic Acid (PNA)–DNA Duplexes: Comparison of Hybridization Affinity between Vertically and Horizontally Tethered PNA Probes

We compare the PNA-DNA duplex hybridization characteristics of vertically tethered and new horizontally tethered PNA probes on solid surfaces. The horizontal 15-mer PNA probe has been synthesized with linker molecules attached at three locations (γ-points) positioned along the PNA backbone that provides covalent attachment of the probe with the backbone aligned parallel to the surface, which is important for DNA hybridization assays that use electric field effect sensors for detection. A radioactive labeled assay and real-time surface plasmon resonance (SPR) biosensor are used to assess the probe surface density, nonspecific binding, and DNA hybridization affinity, respectively, of the new PNA probe configuration. The estimated equilibrium dissociation constants of the horizontally tethered duplex and the vertically tethered duplex are of the same order of magnitude (KD ≈ 5 nM), which indicates a sufficient hybridization affinity for many electronic biosensors that benefit from the horizontal alignment, which minimizes the effects of counterion screening.