Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells
Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells
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DOI：
10.1073/pnas.95.11.6043
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发表时间：
1998-05-26
影响因子：
11.1
通讯作者：
Pederson, T
中科院分区：
文献类型：
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作者：
Politz, JC;Browne, ES;Pederson, T
将荧光素标记的寡脱氧核苷酸（oligodeoxynucleotides，oligos）导入培养的大鼠成肌细胞，用荧光相关光谱（fluorescence correlation spectroscopy，FCS）和荧光漂白后恢复（fluorescence recovery after photobleaching，FRAP）研究了它们在细胞核内的分子运动。FCS显示，大部分核内oligo（dT）（43%）和oligo（dA）（77%）快速移动，扩散系数为4 × 10（-7）cm（2）/s。有趣的是，这种核内寡核苷酸运动的速率类似于它们在水溶液中测量的扩散速率。此外，我们检测到大部分（45%）的核内寡核苷酸（dT），但不是寡核苷酸（dA），以较慢的速度扩散（小于或等于1 × 10（-7）cm（2）/s）。如果在将oligo（dT）引入细胞之前在溶液中与（未标记的）oligo（dA）预杂交，则这种移动较慢的oligo（dT）的量会大大减少，这可能是因为oligo（dT）随后无法与内源性poly（A）RNA杂交。FCS测量的大部分较慢oligo（dT）群体的扩散速率接近与大聚腺苷酸化RNA杂交的oligo（dT）在水溶液中的扩散速率（1.0 x 10（-7）cm（2）/s）。此外，该核内移动速率福尔斯落在计算的平均大小的异质核核糖核蛋白颗粒在水溶液中的扩散速率的范围内。oligo（dT）的亚组分（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移动的速率与水溶液中的速率相当，并进一步表明，这是真实的大核糖核蛋白复合物以及。
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) 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 (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 oligo(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 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.