Characterization of hybridization-sensitive DNA probe by using fluorescence spectroscopy

Abstract

The non-radiative relaxation mechanism of the newly synthesized hybridized-sensitive DNA probe has not been fully understood until now. In this study, the transient dark state of the probe, which is a double fluorescent dye attached to a specific DNA sequence, was investigated using a fluorescence spectroscopy. We showed that radiationless deactivation of the singlet excited state of fluorophores is mainly determined by photo-induced isomerization of the methine bridge by using absorption and fluorescence spectrum results. Radiationless deactivation of the fluorophores is sensitive to the factors that influence the rotation of methine bridge. Experiments done using solutes of differing molecular weight (sucrose, glucose and glycerine) to provide solutions of similar viscosity reveals the importance of solution viscosity and fluorophore conformation on radiationless deactivation. The quantum yield of the fluorophore in high viscous solutions can be explained by a simple diffusion model. This linear relationship is broken when a dimer conformation of the fluorophore starts to form, which in turn influences the emission intensity of the fluorophore. Based on the fluorescence-correlation results, a simplified two energy state model of the probe was constructed and this model provides an insight into the non-radiative relaxation mechanism of the fluorophore and the applications for DNA and RNA detection. The transient dark state was measured in various solvents that are known to affect the intersystem crossing or photoisomerization of the DNA probe. The transient dark state that was measured in a time scale of a few μs is a triplet state and is related to photoisomerization, viscosity, oxygen concentration, and hybridization, all of which are important parameters for cellular microscopy. The transient dark state in a time scale of a sub μs is sensitively changed after addition of target DNA. We can improve the probe’s capability to identify target DNA/RNA by using ...