Dual-color fluorescence fluctuation spectroscopy to study the complexation between poly-l-lysine and oligonucleotides

In this study, dual-color fluorescence fluctuation spectroscopy (FFS) was explored to characterize the association of oligonucleotides (ONs) to cationic polymers. The results from dual-color FFS, in which both the ONs and the cationic polymers were fluorescently labeled, were compared with data obtained from single-color FFS in which only the ONs or the cationic polymers were fluorescently marked. As a model, the association of negatively charged 20-mer ONs to positively charged poly-L-lysine (pLL) was studied. The binding of rhodamine green-labeled ONs (RhGr-ONs) to nonlabeled pLL could be clearly observed in the fluorescence fluctuation profiles. Especially, highly intense fluorescence peaks appeared in the fluorescence fluctuations. A highly intense fluorescence peak was considered to originate from one complex in which a number of RhGr-ONs were present. Complexing nonlabeled ONs to rhodamine green-labeled pLL (RhGr-pLL) also resulted in highly intense fluorescence peaks, indicating not only that the complexes consisted of a number of ONs but also that numerous RhGr-pLL chains were present. Upon complexation of red-labeled pLL (Cy5-pLL) to green-labeled ONs (RhGr-ONs), highly intense fluorescence peaks occurred simultaneously in the red and green detector. These data proved the multimolecular composition of the polyplexes and agreed with the observations from single-color FFS.