Fluorescence Spectroscopy: Application To Characterize The Interaction Between Biological Macromolecules And The Helicase Enzymatic Activity Studies

This thesis presents the applications of fluorescence detection approaches in understanding the fundamental principles of the light activation of biomolecules, bioassemblies, and their catalytic mechanisms. In this context, three frequently used fluorescent methods have been discussed.The first technique, the fluorescence cross-correlation spectroscopy, based on measurements in micro-volumes with weak molecular concentration, has been essentially applied to monitor the cross-correlation of the fluorescence fluctuations of the two complementary DNA strands. In particular, the helicase activity of E.Coli RecQ enzyme and the strand annealing activity of human RecQ5βhelicase have been monitored. Results proved that the FCCS approach is particularly well-suited for monitoring the RecQ helicase enzymatic activity.The second technique, the fluorescence steady-state anisotropy measurements, has been adopted to analyse impact of the two main Raltegravir resistance pathways (N155H and G140S/Q148H) on HIV viral replication and the catalytic properties of recombinant integrase (INs). Results demonstrated the Q148H mutation is responsible for predominant resistance to Raltegravir whereas the G140S mutation increases viral fitness in the context of double mutant G140S/Q148H.The third technique, the time-resolved photoluminescence decay measurement, has been conducted to characterise the fluorescent properties of MPA capped CdTe quantum dots (QDs). Results confirmed the advantages of QDs and their promising applications in fluorescent labelling.In conclusion, this thesis encompasses the fundamentals and various applications involving the integration of light, photonics and biology into biophotonics.