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DNA折纸纳米结构具有很强的多功能性，能够以纳米精度对生物分子进行空间选择性固定，因此越来越多地用于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折纸基板不仅提供了高定位精度，而且由于其相对较高的机械刚性，可以最大限度地减少热波动。因此，DNA折纸底物的结构特性，特别是其稳定性，成为进行此类单分子研究的关键问题。在这个项目中，我们将在分子水平上研究各种混沌剂与不同DNA结构的相互作用。虽然像胍（Gdm+）或四丙基铵（TPA+）盐这样的混乱剂是蛋白质结构的变性剂，经常用于蛋白质展开研究，但它们与DNA的相互作用却鲜为人知。因此，阐明它们攻击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一步。然而，通过这些试剂对DNA折纸变性过程的分子理解，由于在单个DNA折纸中发现的各种自然和非自然DNA结构而进一步复杂化。因此，该项目旨在阐明混乱-DNA相互作用的机制，特别关注DNA结构的作用。这将通过使用不同核苷酸序列的天然DNA、简单的人工DNA纳米结构和复杂的二维和三维DNA折纸纳米结构来实现。后者能够控制诱导过缠绕和欠缠绕的DNA双工结构。原子力显微镜将监测DNA折纸纳米结构的整体形态变化，特别是DNA折纸变性，而光谱学将获得有关混乱物与DNA螺旋分子相互作用的详细信息。利用这种实验方法，Gdm+和TPA+与DNA相互作用中的反离子效应将被研究，这些效应已知在蛋白质变性中起重要作用。此外，为了阐明盐析诱导DNA变性的分子机制，还将讨论水合阳离子的作用。因此，本项目的实验结果将为了解Gdm+和TPA+等向乱剂与核酸的相互作用提供坚实的基础。通过基因组DNA与人工DNA纳米结构的比较，还将阐明迄今为止尚未令人满意的非天然DNA结构的化学特性。