Utilizing RNA aptamers to probe a physiologically important heme-regulated cellular network.
Utilizing RNA aptamers to probe a physiologically important heme-regulated cellular network.
复制标题
利用 RNA 适体探测生理上重要的血红素调节细胞网络。
DOI：
10.1021/cb6002527
复制
发表时间：
2006
影响因子：
4
通讯作者：
Marletta,MichaelA
中科院分区：
文献类型：
--
作者：
Niles,JacquinC;Marletta,MichaelA
广泛适用的策略，促进直接和选择性的调节细胞内水平的生理上重要的小分子是必不可少的解剖其整体和多重作用的细胞过程。因此，我们一直在探索RNA适体用于此目的的适用性。使用大肠杆菌血红素生物合成途径作为负反馈调节过程的简单模型，我们表明，血红素结合RNA适体，在体外开发和细胞内表达，诱导血红素依赖性生长缺陷的大肠杆菌。在将δ-氨基乙酰丙酸（δ-ALA）转化为下游产物方面有缺陷的大肠杆菌血红素营养缺陷型。相对于对照寡核苷酸，适体还在血红素限制条件下生长的细胞中诱导δ-ALA积累。增加培养基中血红素的浓度完全逆转生长缺陷和δ-ALA积累，除了两种适体的逆转是部分的。因此，这些适体特异性地靶向它们的同源配体，并在功能上调节其细胞内浓度，表明RNA适体是用于阐明血红素和可能的其他小分子在调节细胞网络中的作用的有用工具。
Broadly applicable strategies facilitating direct and selective modulation of the intracellular levels of physiologically important small molecules are essential for dissecting their integral and multiple roles in cellular processes. Therefore, we have been exploring the suitability of RNA aptamers for this purpose. Using theEscherichia coliheme biosynthetic pathway as a simple model of a negative feedback regulated process, we show that heme-binding RNA aptamers, developedin vitroand expressed intracellularly, induce a heme-dependent growth defect in anE. coliheme auxotroph defective in converting δ-aminolevulinic (δ-ALA) acid into downstream products. Relative to a control oligonucleotide, the aptamers also induce δ-ALA accumulation in cells grown under heme-limiting conditions. Increasing the concentration of heme in the media completely reverses both the growth defect and δ-ALA accumulation, except for two aptamers for which reversal is partial. Thus, these aptamers specifically target their cognate ligandin vivoand functionally modulate its intracellular concentration, demonstrating that RNA aptamers are useful tools for elucidating the role of heme and possibly other small molecules in regulating cellular networks.