INTRANUCLEAR DIFFUSION AND HYBRIDIZATION STATE OF OLIGONUCLEOTIDES MEASURED BY FLUORESCENCE CORRELATION SPECTROSCOPY IN LIVING CELLS

Fluorescein-labeled oligodeoxynucleotides (oligos) were introduced int o cultured rat myoblasts, and their molecular movements inside the nuc leus were studied by fluorescence correlation spectroscopy (FCS) and f luorescence 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) cm(2)/s. In terestingly, this rate of intranuclear oligo movement is similar to th eir diffusion rates measured in aqueous solution. In addition, we dete cted a large fraction (45%) of the intranuclear oligo(dT), but not oli go(dA), diffusing at slower rates (less than or equal to 1 x 10(-7) cm (2)/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 ol igo(dT) hybridized to a large polyadenylated RNA (1.0 x 10(-7) cm(2)/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 olig o(dT) (15%) moved over 10-fold more slowly, suggesting it was bound to very large macromolecular complexes. Average diffusion coefficients o btained from FRAP experiments were in agreement with the FCS data. The se results demonstrate that oligos can move about within the nucleus a t rates comparable to those in aqueous solution and further suggest th at this is true for large ribonucleoprotein complexes as well.