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

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