Design of nuclease resistant protein kinase Cα DNA enzymes with potential therapeutic application
Design of nuclease resistant protein kinase Cα DNA enzymes with potential therapeutic application
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DOI：
10.1006/jmbi.2000.3491
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发表时间：
2000-02-25
影响因子：
5.6
通讯作者：
Leirdal, M
中科院分区：
文献类型：
--
作者：
Sioud, M;Leirdal, M
对于催化核酸的治疗应用，希望有在生理浓度下具有活性的小的、稳定的和廉价的化合物。我们探索了使用通用的10-23DNA催化核心来抑制恶性细胞中蛋白激酶Cα(PKCα)亚型表达的可能性。通过在PKCA DNA酶中引入3‘-3’-倒置胸苷核苷酸或定点硫代修饰，我们设计了稳定的催化剂，保持了显著的体外切割活性。特别是，在反义臂和催化核心的嘧啶残基中含有硫代磷酸类似物的DNA酶在50%的人血清中非常稳定(t(1/2)>；90小时)，并在纳摩尔浓度下对体外细胞生长抑制高达90%。对PKCα基因表达的抑制是序列特异性的，因为反义臂反向的DNA酶被发现是无效的。荧光显微镜分析表明，5‘-异硫氰酸荧光素标记的DNA酶主要定位在细胞核内。DNA酶处理后的细胞大部分被细胞凋亡所杀死。所描述的PKCαDNA酶触发恶性细胞凋亡(凋亡酶)的能力表明了它们的治疗潜力。此外，这些试剂可能是探索基因功能的有价值的工具。(C)2000年学术出版社。
For the therapeutic application of catalytic nucleic acids it is desirable to have small, stable and inexpensive compounds that are active at physiological Mg2+ concentrations. We have explored the possibility of using the versatile 10-23 DNA catalytic core to suppress the expression of the protein kinase C alpha (PKC alpha) isoform in malignant cells. By introducing either a 3'-3'-inverted thymidine nucleotide or site-specific phosphorothioate modification into a PKCa DNA enzyme, we have designed stable catalysts that retained a significant in vitro cleavage activity. Ln particular, a DNA enzyme containing phosphorothioate analogues in the antisense arms and in the pyrimidine residues of the catalytic core was found to be remarkably stable in 50 % human serum (t(1/2) > 90 hours) and inhibited in vitro cell growth by up to 90 % at nanomolar concentrations. The inhibition of PKC alpha gene expression is sequence-specific, as a DNA enzyme with reversed antisense arms was found to be ineffective. Epifluorescence microscopic analysis of cells transfected with a 5' fluorescein isothiocyanate-conjugated DNA enzyme showed that the DNA enzyme molecules are mainly localised in the nuclei. Most of the DNA enzyme-treated cells were killed by apoptosis. The ability of the described PKC alpha DNA enzymes to trigger apoptosis (apoptozymes) in malignant cells illustrates their therapeutic potential. Furthermore, such agents can be a valuable tool for probing gene function. (C) 2000 Academic Press.