Comparison of cellular binding and uptake of antisense phosphodiester, phosphorothioate, and mixed phosphorothioate and methylphosphonate oligonucleotides.
Comparison of cellular binding and uptake of antisense phosphodiester, phosphorothioate, and mixed phosphorothioate and methylphosphonate oligonucleotides.
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DOI：
10.1089/ard.1993.3.53
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Q. Zhao;S. Matson;C. Herrera;E. Fisher;H. Yu;A. Krieg
中科院分区：
文献类型：
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作者：
Q. Zhao;S. Matson;C. Herrera;E. Fisher;H. Yu;A. Krieg
利用荧光素（FITC）偶联寡核苷酸研究了硫代磷（s -寡核苷酸）或末端硫代磷-磷酸二酯（s -o -寡核苷酸）或甲基膦-磷酸二酯（mp -o -寡核苷酸）修饰对小鼠脾细胞表面结合、摄取和降解的影响。s -寡核苷酸的细胞结合和摄取最高，其次是S-O-寡核苷酸、O-寡核苷酸和mp -O-寡核苷酸。竞争研究表明，s -寡核苷酸对细胞膜寡核苷酸结合位点的亲和力增加，因为它们可以以仅0.1的摩尔比完全阻断o -寡核苷酸的结合。B细胞对所有寡核苷酸的摄取都高于T细胞，并通过B细胞有丝分裂原脂多糖的刺激而增加。虽然我们的细胞已经用常规技术纯化，以消除死细胞，但仍有大约5%的细胞死亡或死亡，这是用碘化丙啶染色的流式细胞术确定的。值得注意的是，寡核苷酸与死细胞的关联比与活细胞的关联大大约50倍。共聚焦显微镜证实活细胞中的寡核苷酸在细胞内，并且在4小时内显示很少的核摄取。虽然细胞内o -寡核苷酸在4小时内明显降解，但没有检测到S-， S- o或mp -o -寡核苷酸的降解。
The effects of phosphorothioate (S-oligonucleotide) or terminal phosphorothioate-phosphodiester (S-O-oligonucleotides) or methylphosphonate-phosphodiester (MP-O-oligonucleotides) modifications on mouse spleen cell surface binding, uptake, and degradation were studied using fluorescein (FITC)-conjugated oligonucleotides. S-oligonucleotides had the highest cell binding and uptake, followed by S-O-, O-, and MP-O-oligonucleotides. Competition studies indicated that S-oligonucleotides have an increased affinity for cell membrane oligonucleotide binding sites, because they could completely block O-oligonucleotide binding at a molar ratio of just 0.1. Uptake of all oligonucleotides was higher in B cells than T cells and was increased by stimulation with the B-cell mitogen, lipopolysaccharide. Although our cells had been purified using conventional techniques to eliminate dead cells, there remained about 5% of cells that were dead or dying, as determined by flow cytometry using propidium iodide staining. Of note, oligonucleotide association with dead cells was approximately 50-fold greater than that with living cells. Confocal microscopy confirmed that the oligonucleotides in living cells were intracellular, and indicated little nuclear uptake by 4 h. While extensive degradation of intracellular O-oligonucleotides was apparent by 4 h, there was no detectable degradation of S-, S-O, or MP-O-oligonucleotides.