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Peptide nucleic acid (PNA), a polyamide DNA mimic, has inspired the development of a variety of hybridization-based methods for the detection, quantification, purification, and characterization of nucleic acids owing to the stability of the PNA/DNA duplex. In this work, PNA probes complementary to a specific sequence of Roundup Ready soybean were immobilized onto a sensor surface via a self-assembled matrix employing streptavidin/biotin binding. The specific hybridization of PNA and DNA has been monitored by applying the chromophore-labeled DNA target oligonucleotides to the PNA modified Au sensor surface in real time using surface plasmon field-enhanced fluorescence spectroscopy. The authors demonstrate three kinds of experiments called global, titration, and kinetic analyses for the determination of rate constants for the association (k(on)) and dissociation processes (k(off)), and the affinity constant (K(A)) of the PNA/DNA duplex formation by fitting the data to a simple Langmuir model. Discrimination of a single base mismatched DNA (15mer) target on a 15mer PNA probe was documented, with a difference of the affinity constant of two orders of magnitude. Finally, the affinity constant for the hybridization of a long polymerase chain reaction product (169mer) obtained by amplification of DNA extracted from genetically modified soybean reference material has been determined by a kinetic-titration analysis. The results show the influence of a Coulomb barrier at high target surface coverage even for the hybridization to PNA at low ionic strength.

Peptide nucleic acids (PNAs) are mimics of oligonucleotides containing a neutral peptidelike backbone and are able to bind complementary DNA targets with high affinity and selectivity. In order to investigate the effect of the ionic strength of the buffer solution, hybridization experiments with PNAs as (catcher) probes and DNAs as target oligonucleotides were performed in different salt solutions. Surface plasmon field-enhanced fluorescence spectroscopy was employed for real-time monitoring of DNA hybridizations to surface bound PNA. Probes with three different strand lengths were immobilized by self-assembly on the sensor surface. By introducing Cy5-labeled DNA targets the affinity constants, K(A)=k(on) (association)/k(off) (dissociation), were determined for fully complementary (MM0) as well as for single base mismatched (MM1) duplexes. Furthermore, the thermal stability of each duplex was determined by measuring melting curves in solution which was then compared to the kinetic and affinity parameters determined for the surface hybridization reactions. The results indicate that ions do not play a significant role for the PNA/DNA hybridization kinetics at surfaces. However, changes in the configuration of the PNA/DNA duplex due to the ionic strength variations influence the fluorescence yield drastically.