Chin D J, Green G A, Zon G, Szoka F C, Straubinger R M
Department of Pharmacology, University of California, San Francisco 94143.
New Biol. 1990 Dec;2(12):1091-100.
The intracellular transport and fate of nucleic acids is poorly understood. To study this process, we injected fluorescent oligodeoxyribonucleotides (oligos) into the cytoplasm of CV-1 epithelial cells and primary human fibroblasts. Rapid nuclear accumulation was found with the phosphodiester (PD), phosphorothioate (PT), and methylphosphonate (MP) forms of a 28-mer oligo complimentary to the rev mRNA of the human immunodeficiency virus type 1. Migration of the oligos in the cytoplasm was slower than diffusion of a coinjected dextran, but the oligos freely diffused into the nucleus. Nuclear incorporation was temperature but not energy dependent. The intranuclear distribution of the oligos was influenced by the chemistry of internucleoside linkages. The PD oligos and, to a lesser extent, the PT oligos colocalized with small nuclear ribonucleoproteins (snRNPs), whereas the MP oligos colocalized with concentrated regions of genomic DNA. These data have important implications for our understanding of the transport and accumulation of exogenous nucleic acids in mammalian nuclei, and the assay described could potentially be used for testing the efficacy of oligos designed as therapeutic agents.
人们对核酸的细胞内运输和归宿了解甚少。为了研究这一过程，我们将荧光寡脱氧核糖核苷酸（oligos）注入CV - 1上皮细胞和原代人成纤维细胞的细胞质中。发现与人类免疫缺陷病毒1型的rev mRNA互补的28聚体oligo的磷酸二酯（PD）、硫代磷酸酯（PT）和甲基磷酸酯（MP）形式能快速积累到细胞核中。oligos在细胞质中的迁移比共注射的葡聚糖的扩散要慢，但oligos能自由扩散到细胞核中。细胞核摄取是温度依赖性的而非能量依赖性的。oligos在细胞核内的分布受核苷间连接化学性质的影响。PD oligos以及在较小程度上PT oligos与小核核糖核蛋白（snRNPs）共定位，而MP oligos与基因组DNA的浓缩区域共定位。这些数据对于我们理解外源核酸在哺乳动物细胞核中的运输和积累具有重要意义，并且所描述的检测方法有可能用于测试设计为治疗剂的oligos的功效。
