Molecular mechanisms of the interaction of chaotropic salts with natural and artificial DNA structures

离液盐与天然和人工 DNA 结构相互作用的分子机制

• 批准号: 428230890
• 负责人: Professor Dr. Karim Fahmy, Ph.D.
• 金额: --
• 依托单位: Helmholtz-Zentrum Dresden-Rossendorf (HZDR)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/428230890?language=en
• 关键词: Molecular; mechanisms; interaction; chaotropic; salts

Due to their great versatility which enables the spatially selective immobilization of biomolecules with nanometer precision, DNA origami nanostructures are increasingly used substrates for the single-molecule investigation of various biochemical and biophysical processes. In these applications, the DNA origami substrates not only provide high positioning accuracy but also minimize thermal fluctuations due to their comparatively high mechanical rigidity. Consequently, the structural properties and especially the stability of the DNA origami substrates have become key issues in conducting such single-molecule studies. In this project, we will investigate the interaction of various chaotropic agents with different DNA structures at a molecular level. While chaotropic agents such as guanidinium (Gdm+) or tetrapropylammonium (TPA+) salts are established denaturants of protein structure and frequently used in protein unfolding studies, less is known about their interaction with DNA. Elucidating the mechanisms of their attack on DNA thus represents an important step toward DNA origami-based single-molecule studies of protein folding and unfolding dynamics. Molecular understanding of the processes involved in DNA origami denaturation by such agents, however, is further complicated by the various natural and non-natural DNA structures found within a single DNA origami. This project therefore aims at elucidating the mechanisms of chaotrope-DNA interactions with a particular focus on the role of DNA structure. This will be achieved by employing natural DNAs of different nucleotide sequence, simple artificial DNA nanostructures, and complex 2D and 3D DNA origami nanostructures. The latter enables the controlled induction of over- and underwound DNA duplex structures. Changes in the overall morphology of the DNA origami nanostructures and in particularly DNA origami denaturation will be monitored by atomic force microscopy while optical spectroscopy will yield detailed information about the molecular interactions of the chaotropes with the DNA helices. Using this experimental approach, counterion effects in the interaction of Gdm+ and TPA+ with DNA will be investigated that are known to play significant roles in protein denaturation. Furthermore, also the effect of hydrated cations will be addressed in order to elucidate the molecular mechanisms of salting-out-induced DNA denaturation.The experimental results of this project will thus provide a solid foundation for developing an understanding of the interaction of chaotropic agents such as Gdm+ and TPA+ with nucleic acids. By comparing genomic DNAs to artificial DNA nanostructures, also the so far only unsatisfyingly explored chemical peculiarities of non-natural DNA structures will be elucidated.

由于它们的多功能性使生物分子具有纳米精度的空间选择性固定，因此DNA折纸纳米结构越来越多地使用了底物，用于对各种生物化学和生物物理过程的单分子研究。在这些应用中，DNA折纸底物不仅提供了高定位精度，而且由于机械刚性相对较高而使热波动最小化。因此，在进行此类单分子研究方面，DNA折纸底物的结构特性，尤其是DNA折纸底物的稳定性已成为关键问题。 在这个项目中，我们将研究各种交际剂与分子水平上不同DNA结构的相互作用。虽然诸如鸟苷（GDM+）或四丙基铵（TPA+）盐之类的混合物剂是蛋白质结构的变性剂，并且经常用于蛋白质展开研究中，但对它们与DNA的相互作用的了解较少。因此，阐明其攻击DNA的机制是朝着基于DNA折纸的蛋白质折叠和展开动力学的单分子研究迈出的重要步骤。然而，这种药物对DNA折纸变性所涉及的过程的分子理解使在单个DNA折纸中发现的各种天然和非天然DNA结构更加复杂。因此，该项目旨在阐明Chaotrope-DNA相互作用的机制，以特别关注DNA结构的作用。这将通过采用不同核苷酸序列，简单人工DNA纳米结构以及复杂的2D和3D DNA折纸纳米结构的天然DNA来实现。后者可以受控诱导过度和内部的DNA双链结构。 DNA折纸纳米结构的总体形态的变化以及特别是DNA折纸变性的变化将通过原子力显微镜监测，而光谱将产生有关交际的分子相互作用的详细信息。使用这种实验方法，将研究对GDM+和TPA+与DNA相互作用的抗衡作用，该效应将研究在蛋白质变性中起着重要作用。此外，还将解决水合阳离子的作用，以阐明盐溶液诱导的DNA变性的分子机制。因此，该项目的实验结果将为建立对诸如GDM+和TPA+与核酸的诸如GDM+和TPA之类的混合物相互作用的相互作用提供稳固的基础。通过将基因组DNA与人工DNA纳米结构进行比较，还将阐明迄今为止唯一不满意的非天然DNA结构的化学特征。