Politz J C, Browne E S, Wolf D E, Pederson T
Worcester Foundation for Biomedical Research, University of Massachusetts Medical Center, Worcester Foundation Campus, Shrewsbury, MA 01545, USA.
Proc Natl Acad Sci U S A. 1998 May 26;95(11):6043-8. doi: 10.1073/pnas.95.11.6043.
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.
将荧光素标记的寡脱氧核苷酸（寡核苷酸）导入培养的大鼠成肌细胞中，并用荧光相关光谱法（FCS）和光漂白后荧光恢复法（FRAP）研究其在细胞核内的分子运动。FCS显示，细胞核内大部分寡聚（dT）（43%）和寡聚（dA）（77%）以4×10⁻⁷cm²/s的扩散系数快速移动。有趣的是，这种细胞核内寡核苷酸的移动速率与其在水溶液中测得的扩散速率相似。此外，我们检测到细胞核内大部分寡聚（dT）（45%），但不是寡聚（dA），以较慢的速率（≤1×10⁻⁷cm²/s）扩散。如果在将寡聚（dT）引入细胞之前，先在溶液中与（未标记的）寡聚（dA）预杂交，那么这种移动较慢的寡聚（dT）的量会大大减少，推测这是因为寡聚（dT）随后无法与内源性多聚（A）RNA进行后续杂交。FCS测量的大部分移动较慢的寡聚（dT）群体的扩散速率接近与大型多聚腺苷酸化RNA杂交的寡聚（dT）在水溶液中的扩散速率（1.0×10⁻⁷cm²/s）。此外，这种细胞核内的移动速率落在水溶液中平均大小的不均一核核糖核蛋白颗粒计算出的扩散速率范围内。一小部分寡聚（dT）（15%）移动速度慢10倍以上，表明它与非常大的大分子复合物结合。从FRAP实验获得的平均扩散系数与FCS数据一致。这些结果表明，寡核苷酸可以在细胞核内以与水溶液中相当的速率移动，并且进一步表明大型核糖核蛋白复合物也是如此。
