Kinetic and affinity analyses of hybridization reactions between peptide nucleic acid probes and DNA targets using surface plasmon field-enhanced fluorescence spectroscopy

Abstract

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 (kon) and dissociation processes (koff, and the affinity constant (KA) 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.

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“…Moreover, the transistor sensor sensitivity was investigated by conducting the control experiments with and with out target DNA injection down to a 1 nM concentration (Figure 2d). This remarkable sensitivity is similar to the sensitivity previously reported using fluorescence based sensors 25…”

“…Amine terminated PNA‐15mers (P2–15mers) were attached to the pMA layer (Scheme 1c), which were used as the selective probe to target DNA. PNA (P2–15mer) probes have been shown to be effective in discriminating between single base differences in applications using short sequences 25, 26. In the presence of an aqueous buffer, the anhydride groups on the polymer surface are hydrolyzed and converted to carboxylic acids, which were activated by circulating a solution containing 0.2 M N ‐ethyl‐ N′ ‐(3‐dimethylaminopropyl)‐carbodiimide hydrochloride (EDC) and 0.05 M N ‐hydroxysuccinimide (NHS) in the flow cell at a constant rate of 300 μL/min for 20 min.…”

“…This effect is also prevalent in the SPFS measurements, but to a lower extent since no gate bias is present. Operating in high ionic strength buffer solutions or diluting the probe density of the sensor matrix could limit the influence of Coulombic repulsion on the hybridization kinetics;25 however, both of these conditions would adversely affect the sensor sensitivity. Moreover, the transistor sensor sensitivity was investigated by conducting the control experiments with and with out target DNA injection down to a 1 nM concentration (Figure 2d).…”

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“…[12,13]. These values are also close to the results obtained by SPFS for an interfacial architecture in which the capture probe layer was surface attached via a biotin-moiety attaching the probe sequence to a straptavidin matrix [14].…”

“…As a consequence, the affinity constants K A =k on /k off decreased by two orders of magnitude in going from a fully matched to a singly mismatched target sequence. This has been seen before with other interfacial architectures, e.g., based on biotinylated probe strands surface-immobilzed via a straptavidin matrix [12]. Within the Langmuir adsorption model, these affinity constants and the resulting K d values can then be compared with those obtained by titration experiments.…”