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

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.