Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells

Abstract

Fluorescein-labeled oligodeoxynucleotides (oligos) were introduced into cultured rat myoblasts, and their molecular movements inside the nucleus were studied by fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP). FCS revealed that a large fraction of both intranuclear oligo(dT) (43%) and oligo(dA) (77%) moves rapidly with a diffusion coefficient of 4 x 10(-7) cm2/s. Interestingly, this rate of intranuclear oligo movement is similar to their diffusion rates measured in aqueous solution. In addition, we detected a large fraction (45%) of the intranuclear oligo(dT), but not oligo(dA), diffusing at slower rates (</=1 x 10(-7) cm2/s). The amount of this slower-moving oligo(dT) was greatly reduced if the oligo(dT) was prehybridized in solution with (unlabeled) oligo(dA) prior to introduction to cells, presumably because the oligo(dT) was then unavailable for subsequent hybridization to endogenous poly(A) RNA. The FCS-measured diffusion rate for much of the slower oligo(dT) population approximated the diffusion rate in aqueous solution of oligo(dT) hybridized to a large polyadenylated RNA (1.0 x 10(-7) cm2/s). Moreover, this intranuclear movement rate falls within the range of calculated diffusion rates for an average-sized heterogeneous nuclear ribonucleoprotein particle in aqueous solution. A subfraction of oligo(dT) (15%) moved over 10-fold more slowly, suggesting it was bound to very large macromolecular complexes. Average diffusion coefficients obtained from FRAP experiments were in agreement with the FCS data. These results demonstrate that oligos can move about within the nucleus at rates comparable to those in aqueous solution and further suggest that this is true for large ribonucleoprotein complexes as well.

Figure 1

Hybridization of oligo(dT) to poly(A) RNA in solution measured by FCS. Aliquots containing 0.4 pmol unlabeled poly(A) RNA (7.5 kb) were added to 20 nM fluorescein-labeled oligo(dT) and, before and after each addition, ten 20-s FCS samplings were performed. (A) Autocorrelation curves (thin lines) obtained from oligo(dT) alone in solution and after last addition of poly(A) RNA. The dashed thick [oligo(dT) alone] and solid thick [hybridized oligo(dT)] lines are the best fit to each autocorrelation curve by using nonlinear least-squares analysis. (B) Histogram showing the extent of hybridization with increasing concentrations of poly(A) RNA. Hatched bars represent the fraction of oligo(dT) moving at 0.23 ms (unhybridized); solid bars represent the fraction of oligo(dT) moving at 1.9 ms (hybridized).

Figure 2

FCS autocorrelation curves of oligo(dT) and oligo(dA) in nuclei. L6 myoblasts were incubated with fluorescein-labeled oligo(dT) or oligo(dA) and the movements of the oligos in nuclei were measured by FCS as described. (A) Autocorrelation curves (thin lines) and best fit curves (obtained by using nonlinear least-squares analysis) for nuclei containing oligo(dT) (thick solid line) or oligo(dA) (thick dashed line). (B) Fluorescein-labeled oligo(dT) was prehybridized in vitro with unlabeled oligo(dA) and then incubated with cells as usual before FCS analysis. The thick dashed line is the best fit curve for the prehybridized oligo(dT) autocorrelation curve (thin line); the solid thick line is the best fit curve for an autocorrelation curve of oligo(dT) (not prehybridized) from the same experiment.

Figure 3

Summary of FCS-determined oligo diffusion rates in L6 nuclei. Multiple FCS readings were performed in nuclei of cells treated with oligo(dT), oligo(dA) or prehybridized oligo(dT) (see Fig. 2), and the data obtained from the best fit for each measurement were sorted into five categories representing various diffusion times. The average fraction of oligo and its average diffusion time within each of the five categories were calculated after weighting each value by using the number of fluorescent particles present in each category (Table 2) and then plotted. (A) Plot showing distribution of oligo(dT) (solid line) compared with oligo(dA) (dashed line). (B) Distribution of oligo(dT) (solid line) compared with prehybridized oligo(dT) (dashed line, see Fig. 2). The vertical bars represent standard error of the mean.