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 f luorescence 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 ؋ 10 ؊7 cm 2 ͞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 ؋ 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 oligo(dT) hybridized to a large polyadenylated RNA (1.0 ؋ 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 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.An understanding of the physical environment inside the cell nucleus is central to a coherent view of gene expression. It is important to know how the viscosity and molecular diffusion rates in the nucleus of a living cell compare with experimental conditions in vitro, where interactions between nucleic acids and proteins are studied at high dilution in aqueous solution. For example, it is not clear whether ribonucleoprotein (RNP) complexes can diffuse freely about the nucleus, impeded only by locally high concentrations of macromolecules or, alternatively, are bound or compartmentalized in such a way as to constrain their motion. Some observations suggest that premRNA transcripts are tethered to elements of the transcriptional, splicing, and͞or polyadenylation machinery (1-3), and it has been proposed that processed mRNAs make their way out of the nucleus by molecular diffusion (4, 5). However, mediated processes have not been ruled out, and the functional relationships between RNA export and nuclear structure remain unclear (6).Recently, Politz et al. (7) characterized nucleic acid uptake and hybridization in living cells by in situ reverse transcription and found that fluorescently labeled oligo(dT) can be taken up by living cells and form hybrid...

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