No enhancement of nuclear entry by direct conjugation of a nuclear localization signal peptide to linearized DNA
No enhancement of nuclear entry by direct conjugation of a nuclear localization signal peptide to linearized DNA
复制标题
DOI：
10.1021/bc034075e
复制
发表时间：
2003-11-01
影响因子：
4.7
通讯作者：
Harashima, H
中科院分区：
文献类型：
--
作者：
Tanimoto, M;Kamiya, H;Harashima, H
外源DNA有效进入细胞核是非病毒载体基因治疗成功的关键因素之一。为了重新解决直接附着在DNA上的核定位信号（NLS）肽的影响，我们制备了三个哑铃形的绿色荧光蛋白（GFP）编码DNA，其中含有一个或两个NLS肽。该肽通过与修饰的尿嘧啶碱基与二氧辛胺连接物之间的交联反应与形成环的寡核苷酸结合，然后将寡核苷酸连接到DNA分子上。将nls结合的DNA哑铃微注射到猿猴COS-7细胞的细胞质和细胞核中。此外，未结合的DNA哑铃，与或不修改尿嘧啶碱基，也进行了比较检查。在细胞内和核内微注射中，GFP基因的表达效率顺序为：未修饰的DNA大于或等于nls修饰的DNA，小于未修饰的碱基修饰的DNA。因此，我们得出结论：(i)一个或两个NLS肽(s)并没有显著改善DNA的核进入，（ii） DNA的化学修饰降低了细胞核内的转录效率或稳定性。
Efficient nuclear entry of exogenous DNA is one of the key factors toward gene therapy success with nonviral vectors. To re-address the effects of a nuclear localization signal (NLS) peptide attached directly to DNA, we prepared three dumbbell-shaped, green fluorescent protein (GFP)-encoding DNAs containing one or two NLS peptides. The peptide was conjugated to the loop-forming oligodeoxyribo-nucleotides by cross-linking reactions between the peptide and a modified uracil base with a dioxaoctylamino linker, and the oligonucleotides were then ligated to the DNA molecules. The NLS-conjugated DNA dumbbells were microinjected into the cytosols and nuclei of simian COS-7 cells. In addition, unconjugated DNA dumbbells, with or without a modified uracil base, were also examined for comparison. The GFP gene was expressed with efficiencies in the order of the unmodified DNA greater than or equal to the NLS-conjugated DNA > the unconjugated DNA with the base modification, with both cytosolic and intranuclear microinjections. Thus, we concluded that (i) one or two NLS peptide(s) did not dramatically improve the nuclear entry of DNA and that (ii) chemical modification of DNA reduced the transcription efficiency or stability in the nucleus.