Studying Pegylated DNA Complexes by Dual Color Fluorescence Fluctuation Spectroscopy

In this study, dual color fluorescence fluctuation spectroscopy (dual color FFS) was explored to characterize the association of oligonucleotides (ONs) to pegylated cationic block copolymers, both diblock poly(ethylene glycol)−poly(ethylenimine) (pEG-pEI) and multiblock pEG-pEI. The resulting polyelectrolyte complexes consist of a core of polyion strands, surrounded by a shell of pEG chains. To study the association of the ONs to pEG-pEI by dual color FFS, the oligonucleotides and the cationic block copolymers were labeled with spectrally different fluorescent dyes. Two alternative approaches were executed to label the pEG-pEI's: the dye was attached either to the pEG segment or to the cationic pEI segment of pEG-pEI. When the (diblock and multiblock) pEG-pEI's were labeled on the pEG segment, dual color FFS on the doubly labeled FITC-pEG-pEI/Cy5-ONs complexes revealed the simultaneous appearance of peaks of high fluorescence intensity in both detector channels. This indicates that numerous Cy5-ONs and numerous FITC-pEG-pEI chains move together through the detection volume of the FFS instrument, proving the interaction between many Cy5-ONs and many FITC-pEG-pEI strands to form one multimolecular complex. However, when the fluorescent dye was bound to the cationic pEI segment of diblock or multiblock pEG-pEI, these simultaneously occurring peaks were never observed. Gel electrophoresis experiments proved that diblock pEG-pEI labeled at the pEI segment could not complex the ONs anymore. Multiblock pEG-pEI labeled at the pEI segment was still able to fully complex the oligonucleotides (as shown by gel electrophoresis). The absence of peaks of high fluorescence intensity in FFS measurements was attributed to the quenching of the fluorophores in the core of the complexes. From these results, it could be concluded that dual color FFS allows studying the association and dissociation of pEG-pEI/ONs provided that the fluorescent label of the polymer is attached to the pEG segment and not to the pEI segment.