Thermodynamics of Peptide Nucleic Acid Interactions with DNA
Peptide Nucleic Acid (PNA) is a DNA mimic with the natural nucleobases attached to a charge-neutral pseudopeptide backbone. It has remarkably strong and sequence-specific binding affinity to natural nucleic acids (DNA and RNA), mainly attributed to the lack of charge in the PNA backbone.
The ionic effects on the thermal stability of 10 and 15 base pairs long PNA-DNA duplexes have been studied. At low ionic strength (< 0.5 M), increasing salt (Na+) concentration was found to slightly destabilize the PNA-DNA hybrid, whereas DNA-DNA was strongly stabilized, and the uncharged PNA-PNA essentially unaffected. At high ionic strength (> 1 M), increased salt concentration decreased the stability of the three kinds of duplexes in a similar fashion.
By measuring the fluorescence energy transfer (FET) efficiency from fluorescein to rhodamine labels attached to opposite ends of 10-mer PNA-PNA, PNA-DNA, and DNA-DNA duplexes, the estimated end-to-end distances were all found to be consistent with relatively extended duplex conformations. While quite useful for structural characterization, due to label-duplex interactions, FET was found to be less suitable than absorption hyperchromicity for thermodynamic analysis.
A systematic study of the thermodynamics of PNA-DNA duplexes established a length- and sequence-averaged binding constant of 14 M-1bp-1 at 25°C, while the stability of single mismatches in mixed sequence 9-12-mers was found to vary significantly and depend on both stability and nature of the surrounding base pairs. On average, one mismatch turned out to cost as much as the gain of two perfect base pairs. Further analysis of the thermodynamic parameters revealed that strong enthalpy-entropy compensation is important for PNA-DNA interactions.
Based on the thermodynamic data, the hybridization properties of PNA versus a hypothetical genome target was modeled using a simple statistical approach. Though simple, the model gives general insight into sequence-specific binding, and should be applicable to a variety of situations, ranging from gene-targeting to biotechnical applications such as gene-diagnostics.
In addition, FET was also used for kinetic investigation of interactions between structured, naturally occurring regulatory RNA pairs. The overall kinetic rate constants for complex formation of wild-type and mutated pairs were obtained, as well as a preliminary characterization of the early interaction event using the fluorescent nucleobase analog 2-aminopurine.
fluorescence energy transfer