Liu Yingxun, Quan Fusheng, Wang Jinke, Bai Xueyao
College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China.
Sheng Wu Gong Cheng Xue Bao. 2010 Dec;26(12):1683-9.
To investigate the effect of the localization of oligonucleotides decoy (ODNs decoy) on the activation of nuclear factor-kappaB (NF-kappaB) in TNF-alpha induced HeLa cells. The mercapto group-modified nuclear localization signal (NLS) peptide was covalently conjugated to amino group-modified NF-kappaB ODNs decoy by Sulfo-SMCC cross-linker. The NLS-ODNs decoy was transfected into HeLa cells by TransME transfection reagent. The intracellular distribution of fluorescent labeled NLS-ODNs decoy was detected with a microscope. The cell viability was detected by MTT assay, and then the activity of NF-kappaB in cell nuclear extract was assayed by electrophoretic mobility shift assay (EMSA). The results showed that NLS peptide was successfully conjugated to ODNs decoy by Sulfo-SMCC cross-linker. The NLS-ODNs decoy effectively entered into nucleus with high rate of 17.9%. It was observed that the cell viability of HeLa cell was not significantly affected by the transfection of NLS-ODNs decoy, while NLS-ODNs decoy significantly inhibited the activation of NF-kappaB in TNF-alpha induced HeLa cells nuclear extracts. This experiment can provide a new covalent conjugation of NLS peptide to ODNs can effectively drive decoy into nucleus, and thus improve its inhibitory effects on the activation a transcription factor.
探讨寡核苷酸诱饵（ODNs诱饵）的定位对肿瘤坏死因子-α（TNF-α）诱导的HeLa细胞中核因子-κB（NF-κB）激活的影响。通过磺基琥珀酰亚胺基-4-(N-马来酰亚胺甲基)环己烷-1-羧酸酯（Sulfo-SMCC）交联剂将巯基修饰的核定位信号（NLS）肽与氨基修饰的NF-κB ODNs诱饵共价偶联。通过TransME转染试剂将NLS-ODNs诱饵转染至HeLa细胞中。用显微镜检测荧光标记的NLS-ODNs诱饵的细胞内分布。通过MTT法检测细胞活力，然后通过电泳迁移率变动分析（EMSA）检测细胞核提取物中NF-κB的活性。结果显示，通过Sulfo-SMCC交联剂成功将NLS肽与ODNs诱饵偶联。NLS-ODNs诱饵以17.9%的高比率有效进入细胞核。观察到NLS-ODNs诱饵转染对HeLa细胞的细胞活力无显著影响，而NLS-ODNs诱饵显著抑制TNF-α诱导的HeLa细胞核提取物中NF-κB的激活。本实验可为NLS肽与ODNs的新型共价偶联提供依据，其可有效驱使诱饵进入细胞核，从而提高其对转录因子激活的抑制作用。
