Structure and dynamics of an archetypal DNA nanoarchitecture revealed via cryo-EM and molecular dynamics simulations

Abstract

DNA can be folded into rationally designed, unique, and functional materials. To fully realise the potential of these DNA materials, a fundamental understanding of their structure and dynamics is necessary, both in simple solvents as well as more complex and diverse anisotropic environments. Here we analyse an archetypal six-duplex DNA nanoarchitecture with single-particle cryo-electron microscopy and molecular dynamics simulations in solvents of tunable ionic strength and within the anisotropic environment of biological membranes. Outside lipid bilayers, the six-duplex bundle lacks the designed symmetrical barrel-type architecture. Rather, duplexes are arranged in non-hexagonal fashion and are disorted to form a wider, less elongated structure. Insertion into lipid membranes, however, restores the anticipated barrel shape due to lateral duplex compression by the bilayer. The salt concentration has a drastic impact on the stability of the inserted barrel-shaped DNA nanopore given the tunable electrostatic repulsion between the negatively charged duplexes. By synergistically combining experiments and simulations, we increase fundamental understanding into the environment-dependent structural dynamics of a widely used nanoarchitecture. This insight will pave the way for future engineering and biosensing applications.

Dominant Analytical Techniques in DNA Nanotechnology for Various Applications

“…This technique was developed in 1974 with the hope of being able to image biological specimens with all of their water preserved, and keeping it cold enough to prevent evaporation was the driving force . That being said, Cryo-EM is an excellent characterization method for DNA nanostructures, as in many cases, geometry of the nanostructure determines function, and it is essential to ensure that the DNA construct has formed correctly . When applying Cryo-EM to DNA nanostructures, this technique is mainly used for constructs that are developed with a biological application in mind, but it is certainly relevant for any downstream application. , It is as a structural validation technique for 3D wireframe structures designed using tools such as DAEDALUS and also to validate the hierarchical assembly of DNA polyhedra from smaller DNA tiles, both of which are challenging to visualize using AFM and TEM due to their 3D nature and lower DNA density, respectively. , As an additional example, researchers set a goal of inserting a DNA origami nanostructure into a simian virus 40 capsid (SV40).…”

Functional Nanopores Enabled with DNA

“…[152] The assembly pathway of the six-helix bundle pore was recently monitored by ion mobility mass spectrometry, [153] while their structural conformations have been investigated using cryo-electron microscopy and molecular dynamics simulations. [154][155][156] The dimensions of DNA pore structures were further reduced by Göpfrich et al, with the design of a DNA channel comprising solely four duplexes that enclosed a subnanometer lumen (Figure 5f). [157] Using the DNA tile design, the DNA channel was built out of 8 interconnected DNA strands enclosing a lumen of approximately 0.8 nm.…”

“…[179] Furthermore, DNA nanocarriers allow the identification of RNA isoforms by specifically binding target RNA followed by single-molecule pore sensing. [180] Future developments on DNA modification and DNA nanotechnology and nanopore techniques could yield advanced analytical platforms [11] with precisely tuned pores to detect molecular analytes, single-molecule research tools for ligand-receptor binding [104] and chemical/enzymatic reactions, [181] and biomimetic nanostructures [182] to replicate transmembrane flux, [154] signal transduction, and molecular motors. [183]…”

Role of Protonation States in the Stability of Molecular Dynamics Simulations of High-Resolution Membrane Protein Structures

“…To complement cryo-EM structures, molecular dynamics (MD) simulations are often performed. − These help in studying the dynamics of the protein and the solvent as well as the binding and unbinding of lipids, ligands, and ions. The approach of combining structural data with MD simulations is a powerful technique to understand protein structure and function.…”