Fluorescence measurement of hybridization between quencher (DABCYL) labelled PNA probes and a fluoresceine labelled DNA using the Fluorescence BioMelt Package

Significance of the Topic

Reversible hybridization of nucleic acids underpins essential biological processes such as replication, transcription and translation. Monitoring the interaction between peptide nucleic acid (PNA) probes and DNA targets through fluorescence thermal melting offers high sensitivity, specificity and the ability to work at low concentrations. The robustness of PNA probes, their resistance to enzymatic degradation and their strong binding properties make them ideal tools for diagnostic assays and research applications in biomedical and environmental analysis.

Study Objectives and Overview

This application note describes the use of the Agilent Fluorescence BioMelt Package to measure thermal melting profiles of DNA–PNA duplexes. Two lengths of quencher-labelled PNA probes (9mer and 13mer labelled with DABCYL) were hybridized to a 5′ fluorescein-labelled DNA strand. The goals were to determine melting temperatures (Tm) for each duplex, compare stability as a function of PNA length, and demonstrate the value of fluorescence-based thermal melt assays for optimizing hybridization conditions.

Methodology and Instrumentation

The experiment used the Agilent Fluorescence BioMelt system comprising:

• Cary Eclipse Fluorescence Spectrophotometer
• Multicell Peltier temperature block and controller
• Quartz 10 mm cuvettes with stoppers
• Eclipse Thermal Software (Bio software package)

Sample preparation and measurement parameters:

• DNA and PNA probes at 50 nM in 1 mL buffer (100 mM NaCl, 10 mM KPO4, pH 7.1)
• DNA labelled with 5′-6-carboxyfluorescein; PNA labelled with DABCYL
• Excitation at 495 nm, emission detection at 518 nm, PMT voltage 600 V
• Thermal ramps from 20 °C to 95 °C and back at 0.5 °C/min with 5 min holds at extremes
• Tm was calculated by the first-derivative method in the Eclipse Thermal Software

Main Results and Discussion

The 9mer PNA–DNA duplex exhibited a melting temperature of 49.94 °C, while the 13mer duplex melted at 63.9 °C. The higher Tm for the longer PNA reflects increased base stacking and more extensive polyamine backbone interactions compared to DNA–DNA duplexes. As temperature rises, dissociation of the quencher from the fluorescein results in an increase in fluorescence signal, enabling precise determination of melting transitions. These results confirm that PNA probe length significantly influences the thermal stability of the hybrid.

Benefits and Practical Applications

Fluorescence thermal melting using PNA probes offers:

• High sensitivity for low-concentration assays
• Resistance to protease and nuclease degradation in complex samples
• Rapid and reproducible determination of probe–target stability
• Potential for multiplexed detection via different fluorophore–quencher pairs

Such assays are valuable for microbial detection in food, water and environmental monitoring, for designing sequence-specific diagnostics in pharmaceutical research, and for studying nucleic acid interactions in fundamental research.

Future Trends and Potential Applications

Emerging directions include:

• Integration of multi-color FRET assays for simultaneous detection of multiple targets
• Application of rapid thermal melt profiling for RNA and protein conformational analyses
• Automation of assay optimization through advanced software-driven thermodynamic calculations
• Development of PNA-based delivery vehicles for antisense and gene regulation studies

Conclusion

Fluorescence thermal melting assays using DABCYL-labelled PNA probes and fluorescein-labelled DNA provide a powerful method to characterize hybrid stability and to optimize assay conditions. The observed Tm differences between 9mer and 13mer PNA probes illustrate how probe length and backbone chemistry control binding strength. The Agilent Fluorescence BioMelt Package delivers precise temperature control, sensitive fluorescence detection, and automated thermodynamic analysis, making it an effective platform for nucleic acid assay development.

References

• Plum GE, Pilch DS, Singleton SF, Breslauer KJ. Nucleic Acid Hybridization: Triple Stability and Energetics. Annu Rev Biophys Biomol Struct. 1995;24:319-350.
• Boston Probes. PNA Technology Overview. www.bostonprobes.com.
• Guzzo-Pernell N, Tregear GW. Triple Helical DNA Analysis with Hydrophobic Oligonucleotide-Peptide Molecule. Aust J Chem. 2000;53:699-705.
• Guzzo-Pernell N, Lawlor JM, Haralambidis J. Triple Helical DNA Studies. Biomed Pept Proteins Nucleic Acids. 1997;2:107-122.
• Yang M, Ghosh SS, Millar P. Thermodynamics and Kinetics of DNA Triple Helix Formation by Fluorescence. Biochemistry. 1994;33:15329-15337.
• Goforth S. PNA Probe Technology. The Scientist. 2000;14(22):19.
• Agilent Technologies. Part Numbers for Cary Eclipse and Accessories. Agilent Publication SI-A-1833. 2011.
• Morrison LE, Stols LM. Sensitive Fluorescence-Based Thermodynamic and Kinetic Measurements of DNA Hybridization. Biochemistry. 1993;32:3095-3104.
• Breslauer KJ. Thermodynamic Data for Biochemistry and Biotechnology. In: Hinz HJ, ed. Springer-Verlag; 1986:402-427.
• Marky LA, Breslauer KJ. Calculating Thermodynamic Data from Melting Curves. Biopolymers. 1987;26:1601-1620.
• Bush CA. Nucleic Acid Chemistry. In: Ts’o POP, ed. Academic Press; 1974:91-169.
• Patel DJ, Pardi A, Itakura K. DNA Conformation and Dynamics in Solution. Science. 1982;216:581-590.
• Clegg RM. Fluorescence Resonance Energy Transfer and Nucleic Acids. Methods Enzymol. 1992;211:353-388.
• Yaron A, Carmel A, Katchalski-Katzir E. Intramolecularly Quenched Fluorogenic Substrates for Hydrolytic Enzymes. Anal Biochem. 1972;95:228-235.
• Fiandaca MJ, Hyldig-Nielsen JJ, Gildea BD, Coull JM. Self Reporting PNA/DNA Primers for PCR Analysis. Genome Res. 2001;11:609-613.