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Interaction of fluorescently labeled pyrrole-imidazole polyamide probes with fixed and living murine and human cells
Biochimie ( IF 3.3 ) Pub Date : 2018-04-05
Karine Nozeret, François Loll, Gildas Mouta Cardoso, Christophe Escudé, Alexandre S. Boutorine

Pericentromeric heterochromatin plays important roles in controlling gene expression and cellular differentiation. Fluorescent pyrrole-imidazole polyamides targeting murine pericentromeric DNA (major satellites) can be used for the visualization of pericentromeric heterochromatin foci in live mouse cells. New derivatives targeting human repeated DNA sequences (α-satellites) were synthesized and their interaction with target DNA was characterized. The possibility to use major satellite and α -satellite binding polyamides as tools for staining pericentromeric heterochromatin was further investigated in fixed and living mouse and human cells. The staining that was previously observed using the mouse model was further characterized and optimized, but remained limited regarding the fluorophores that can be used. The promising results regarding the staining in the mouse model could not be extended to the human model. Experiments performed in human cells showed chromosomal DNA staining without selectivity. Factors limiting the use of fluorescent polyamides, in particular probe aggregation in the cytoplasm, were investigated. Results are discussed with regards to structure and affinity of probes, density of target sites and chromatin accessibility in both models.



中文翻译:

荧光标记的吡咯-咪唑聚酰胺探针与固定的和活的鼠和人细胞的相互作用

周生大分子异染色质在控制基因表达和细胞分化中起重要作用。靶向鼠腹膜着丝粒DNA(主要卫星)的荧光吡咯-咪唑聚酰胺可用于可视化活小鼠细胞中的腹膜着丝粒异染色质病灶。合成了针对人类重复DNA序列(α-卫星)的新衍生物,并表征了它们与靶DNA的相互作用。在固定的和活的小鼠和人类细胞中,进一步研究了使用主要的卫星和与α卫星结合的聚酰胺作为染色质着丝粒异染色质的工具的可能性。进一步表征和优化了先前使用小鼠模型观察到的染色,但在可使用的荧光团方面仍然有限。关于小鼠模型中的染色的有希望的结果不能扩展到人类模型。在人类细胞中进行的实验显示染色体DNA染色没有选择性。研究了限制使用荧光聚酰胺的因素,特别是探针在细胞质中的聚集。在两个模型中都讨论了有关探针的结构和亲和力,靶位点的密度和染色质可及性的结果。

更新日期:2018-04-06
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