Epigenetic modification, dehydration, and molecular crowding effects on the thermodynamics of i-motif structure formation from C-rich DNA.
Epigenetic modification, dehydration, and molecular crowding effects on the thermodynamics of i-motif structure formation from C-rich DNA.
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DOI：
10.1021/bi401523b
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发表时间：
2014-03-18
期刊：
影响因子：
2.9
通讯作者：
Wadkins, Randy M.
中科院分区：
文献类型：
--
作者：
Bhaysar-Jog, Yogini P.;Van Dornshuld, Eric;Brooks, Tracy A.;Tschumper, Gregory S.;Wadkins, Randy M.
DNA序列具有形成I-基序(IMS)和G-四链(G4S)等二级结构的潜力，在几个癌基因的启动子中含量丰富，在某些情况下，已知调节基因的表达。最近，形成Im的DNA链也被用作纳米设备的功能单元，从药物输送系统到纳米电路。为了理解IMS的基因调控机制以及如何在纳米技术应用中更有效地利用它们，有必要对控制其构象状态和稳定性的因素有一个透彻的了解。以往研究IMS构象动力学的大部分工作都是通过形成Im的合成结构，如串联(CCT)n重复和在标准稀缓冲液体系中完成的。在这里，我们系统地研究了表观遗传修饰、分子拥挤和水化程度对来自c-myc基因启动子的Im形成序列的稳定性的影响。我们的结果表明，胞嘧啶的5-羟甲基化破坏了IMS的稳定性，使其不受温度和pH的影响；相反，5-甲基胞嘧啶的修饰则稳定了IMS。在分子拥挤条件下(PEG300，40%w/v)，微球的热稳定性提高了10℃，∼由6.1±0.1提高到7.0±0.1。最后，Im在1，2-二甲氧基乙烷、2-甲氧基乙醇、乙二醇、1，3-丙二醇和甘油共溶剂中不同程度水化的稳定性表明，由于折叠时水分子的释放，Im通过脱水而稳定。我们的结果强调了考虑表观遗传修饰、分子拥挤和水化程度对Im结构动力学影响的重要性。例如，在IMS中加入5-甲基胞嘧啶和5-羟甲基胞嘧啶可用于微调Im依赖于pH或温度的折叠/去折叠。IMS水化程度的变化还可以提供对IMS折叠/未折叠状态的额外控制，而不必改变周围基质的pH。
DNA sequences with the potential to form secondary structures such as i-motifs (iMs) and G-quadruplexes (G4s) are abundant in the promoters of several oncogenes and, in some instances, are known to regulate gene expression. Recently, iM-forming DNA strands have also been employed as functional units in nanodevices, ranging from drug delivery systems to nanocircuitry. To understand both the mechanism of gene regulation by iMs and how to use them more efficiently in nanotechnological applications, it is essential to have a thorough knowledge of factors that govern their conformational states and stabilities. Most of the prior work to characterize the conformational dynamics of iMs have been done with iM-forming synthetic constructs like tandem (CCT)n repeats and in standard dilute buffer systems. Here, we present a systematic study on the consequences of epigenetic modifications, molecular crowding, and degree of hydration on the stabilities of an iM-forming sequence from the promoter of the c-myc gene. Our results indicate that 5-hydroxymethylation of cytosines destabilized the iMs against thermal and pH-dependent melting; contrarily, 5-methylcytosine modification stabilized the iMs. Under molecular crowding conditions (PEG-300, 40% w/v), the thermal stability of iMs increased by ∼10 °C, and the pKa was raised from 6.1 ± 0.1 to 7.0 ± 0.1. Lastly, the iM’s stability at varying degrees of hydration in 1,2-dimethoxyethane, 2-methoxyethanol, ethylene glycol, 1,3-propanediol, and glycerol cosolvents indicated that the iMs are stabilized by dehydration because of the release of water molecules when folded. Our results highlight the importance of considering the effects of epigenetic modifications, molecular crowding, and the degree of hydration on iM structural dynamics. For example, the incorporation of 5-methylycytosines and 5-hydroxymethlycytosines in iMs could be useful for fine-tuning the pH- or temperature-dependent folding/unfolding of an iM. Variations in the degree of hydration of iMs may also provide an additional control of the folded/unfolded state of iMs without having to change the pH of the surrounding matrix.
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