Ionic and structural specificity effects of natural and synthetic polyamines on the aggregation and resolubilization of single-, double-, and triple-stranded DNA
Ionic and structural specificity effects of natural and synthetic polyamines on the aggregation and resolubilization of single-, double-, and triple-stranded DNA
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DOI：
10.1021/bi9825753
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发表时间：
1999-03-23
期刊：
影响因子：
2.9
通讯作者：
Thomas, TJ
中科院分区：
文献类型：
--
作者：
Saminathan, M;Antony, T;Thomas, TJ
DNA缩合、沉淀和聚集是涉及多价阳离子存在下的DNA-DNA相互作用的相关现象，并研究了它们在细胞DNA包装中的潜在意义。最近的研究表明，凝聚/聚集是细胞摄取DNA用于基因治疗的先决条件。为了阐明DNA聚集的离子和结构因素，我们用离心法、T-m测量和圆二色谱研究了在四价多胺精胺存在下，高相对分子质量和超声处理的小牛胸腺DNA、两个治疗性寡核苷酸和聚(Da).2聚(Dt)三倍体DNA的沉淀和拆分。精胺诱导DNA沉淀的能力依次为三链DNA、双链DNA和GT、单链DNA。相反，它们在高多胺浓度下的分解顺序是相反的。精胺沉淀DNA的有效浓度随培养液中Na+浓度的增加而增加。T-m数据表明，即使在溶解状态下，精胺仍具有稳定DNA的作用。圆二色谱显示了一系列双链和三链DNA的构象变化，在高浓度(约200 mM)的精胺作用下，双链形式恢复了B-DNA的构象。然而，三倍体DNA在溶解状态下仍保持Psi-DNA构象。亚精胺和精胺类似物在沉淀和拆分超声提取的小牛胸腺DNA过程中表现出化学结构特异性效应，精胺的N-4-甲基取代和亚氨基的七甲撑分离分别与亚精胺和精胺的沉淀能力有最大差异。与精胺相比，具有重要治疗意义的双(乙基)取代降低了类似物的沉淀能力。多胺的阳离子效应明显，其中五胺比四胺和三胺更有效，这些结果为揭示多胺沉淀DNA的机制提供了新的见解，并提示了多胺结构在开发用于治疗的基因输送载体方面的重要性。
DNA condensation, precipitation, and aggregation are related phenomena involving DNA-DNA interactions in the presence of multivalent cations, and studied for their potential implications in DNA packaging in the cell. Recent studies have shown that the condensation/aggregation is a prerequisite for the cellular uptake of DNA for gene therapy applications. To elucidate the ionic and structural factors involved in DNA aggregation, we studied the precipitation and resolubilization of high molecular weight and sonicated calf thymus DNA, two therapeutic oligonucleotides, and poly(dA).2Poly(dT) tripler DNA in the presence of the tetravalent polyamine spermine using a centrifugation assay, T-m measurements, and CD spectroscopy. The ability of spermine to provoke DNA precipitation was in the following order: tripler DNA, duplex DNA > single-stranded DNA. In contrast, their resolubilization at high polyamine concentrations followed a reverse order. The effective concentration of spermine to precipitate DNA increased with Na+ in the medium. T-m data indicated the DNA stabilizing effect of spermine even in the resolubilized state. CD spectroscopy revealed a series of sequential conformational alterations of duplex and tripler DNA, with the duplex form regaining the B-DNA conformation at high concentrations (similar to 200 mM) of spermine. The tripler DNA, however, remained in a Psi-DNA conformation in the resolubilized state. Chemical structural specificity effects were exerted by spermidine and spermine analogues in precipitating and resolubilizing sonicated calf thymus DNA, with N-4-methyl substitution of spermidine and a heptamethylene separation of the imino groups of spermine having the maximal difference in the precipitating ability of the analogues compared to spermidine and spermine, respectively. Therapeutically important bis(ethyl) substitution reduced the precipitating ability of the analogues compared to spermine. The effect of the cationicity of polyamines was evident with the pentamines being much more efficacious than the tetramines and triamines, These results provide new insights into the mechanism of DNA precipitation by polyamines, and suggest the importance of polyamine structure in developing gene delivery vehicles for therapeutic applications.