Dynamics of Large Semiflexible Chains Probed by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy was used to probe the dynamics of $\lambda$-phage DNA in aqueous solution labeled with the randomly intercalating dye TOTO. The linear macromolecules (i) carry more than one chromophore and (ii) are larger than the waist of the focal volume. The correlation function decays significantly faster than expected for a stiff globule of corresponding size but is in good agreement with the dynamic model of semiflexible chains including hydrodynamic interactions. As the chromophore density is lowered the correlation time decreases in accordance with this model.

Figure 1

The measured autocorrelation curve $g_2$ for dilute $\lambda$-phage DNA deviates from the correlation function expected for a stiff finite-size polymer with $R_{\mathrm{g}}=0.73\mu \mathrm{m}$ (dashed line). The solid line shows the poor agreement with a best fit to the correlation function of a pointlike particle. The drawings illustrate labeled DNA as a chain of linked chromophores for (a) high and (b) low average label densities.

Figure 2

Comparison of experimental correlation curves with the hydrodynamic model, corresponding to DNA label densities of $1/150$, $1/500$, $1/1000$, and $1/8000{\mathrm{b}\mathrm{p}}^{-1}$ (top to bottom). The solid lines represent fits to Eq. (6). (b) Demonstration of residuals (from third data set). (c) Fitted dynamic contributions $g_{\mathrm{d}\mathrm{i}\mathrm{f}\mathrm{f}}$ (solid line), $\chi$ (dashed line), and $g_{\mathrm{t}\mathrm{r}}$ (dotted line) for the third data set. The functions are offset for presentational purposes.

Figure 3

(a) Diffusion time ${\tau }_{\mathrm{C}}$ vs average label distance, $\Delta$. The solid line indicates the semiflexible chain model, the dashed line the Zimm model. (b) Measured stretching exponent $\beta$ vs $\Delta$.

Figure 4

(a) Number of molecules $N$ per focal volume vs the average label distance, $\Delta$. (b) Reciprocal off rates, $k_{\mathrm{o}\mathrm{f}\mathrm{f}}^{-1}$ vs $\Delta$. The straight line represents a slope of 2.