Controlling Assembly and Activity of Biomimetic DNA Nanopores

Biological channels control the transport of vital biomolecular cargo across the cellular membrane. Reflecting the channels’ critical role, replicating their structure and improving on their function is of significant biomedical and scientific interest. However, de novo design of pores with the typical biological building material of polypeptides is challenging. DNA, by contrast, offers unrivalled structural control due to the simplicity and specificity of Watson-Crick base-pairing. Taking advantage of these properties, DNA membrane pores have been rationally designed with tuneable dimensions. The overall aim of this thesis is to advance on the simple barrel-like synthetic pores and create higher-order function to control pore formation and transport by means of exogenous triggers. The first aim of this thesis was the development of a model system to probe DNA hybridisation under steric constraints. By exploring the effect of DNA hybridisation under steric constraints, such as at membranes and on DNA nanostructures, greater insight was provided for the design of DNA nanopore that assemble in situ and respond to exogenous triggers. The second aim was to design a DNA nanopore that would mimic protein pore formation by undergoing triggered assembly. To transition the inactive pre-pore monomers to an active membrane-spanning oligomeric pore, the locked monomers can be unlocked in the presence of keys to trigger pore assembly. The pore advances functional DNA nanotechnology and synthetic biology by imparting targeted selectivity for pore activity and by serving as a synthetic mimic. The third aim was to build a DNA nanopore that functions as a synthetic protein-gate, allowing the transport of molecular cargo only in the presence of a target exogenous trigger. To function as a protein-gate, a DNA nanopore was designed with a lid featuring an aptamer sequence. In the rational designed structure, the binding of a target protein to the aptamer actuates the lid to open the pore into a transport-active state. A pore with such selective control could then be used in targeted drugdelivery

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

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

Triggered Assembly of a DNA-Based Membrane Channel

Chemistry is in a powerful position to synthetically replicate biomolecular structures. Adding functional complexity is key to increase the biomimetics' value for science and technology yet is difficult to achieve with poorly controlled building materials. Here, we use defined DNA blocks to rationally design a triggerable synthetic nanopore that integrates multiple functions of biological membrane proteins. Soluble triggers bind via molecular recognition to the nanopore components changing their structure and membrane position, which controls the assembly into a defined channel for efficient transmembrane cargo transport. Using ensemble, single-molecule, and simulation analysis, our activatable pore provides insight into the kinetics and structural dynamics of DNA assembly at the membrane interface. The triggered channel advances functional DNA nanotechnology and synthetic biology and will guide the design of controlled nanodevices for sensing, cell biological research, and drug delivery

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