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Background: Fluorescence correlation spectroscopy (FCS) can be used for the determination of diffusion coefficients of single molecules. Since diffusion coefficients are correlated with size and shape of the labeled species, FCS provides information on conformational changes in plasmids aggregates. Methods: A 10-kbp plasmid stained with PicoGreen® was condensed by spermine or liposomes formulated from cationic lipid and egg phosphatidylcholine. Results: The diffusion coefficient of DNA increases from 1.0 × 10–12 m2/s to 3.2 × 10–12 m2/s by the addition of spermine, whereas the addition of cationic liposomes leads to complexes characterized by diffusion coefficients with values ranging from 1.7 to 1.9 × 10–12 m2/s. Conclusions: FCS experiments allow determining the diffusion coefficients of DNA-containing aggregates which provide information regarding the topology and homogeneity of the aggregate. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

We demonstrate that fluorescence correlation spectroscopy (FCS) can be employed to follow the conformational changes of DNA molecules induced by the addition of a cationic condensing compound (spermine). In our experiments the plasmid pHbetaAPr-1-neo (10 kbp; contour length 3.4 microm) was labeled with propidium iodide (PrIo) and then titrated with spermine to induce its condensation. When spermine was applied at concentrations above 5 microM (spermine/DNAphosphate=0.375), the diffusion time of the labeled plasmid dropped from 15 ms down to 3 ms (its diffusion coefficient, D, increased from 1.0x10(-12) m2/s to 6.0x10(-12) m2/s). The application of spermine was also accompanied by decreasing count rate and particle number, reflecting the dye's dissociation. The data presented show that FCS may become a valuable tool in studying supramolecular aggregate formation, especially when association is followed by a change in the hydrodynamic size of the resulting complex.

The presented data show that the FCS technique can be used to detect the DNA condensation process induced with the cationic compound hexadecyltrimethylammonium bromide (HTAB). We have shown that HTAB induces plasmid condensation upon interaction with it. Condensation can be considered to be complete when the diffusion constant reaches its maximum. The HTAB induced increase in diffusion time does not correlate well with the changes observed when count rate and particle number are considered. This observation contradicts data published for another cationic agent, spermine. This apparent discrepancy proves that the mechanisms of interaction between these compounds and DNA are different. Consequently, the different characters of the plots of count rate, diffusion time, and particle number versus condensing agent concentration can be a source of additional information about the nature of cationic compound-DNA interaction.