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.

A Biomimetic DNA-Based Membrane Gate for Protein-Controlled Transport of Cytotoxic Drugs.

Chemistry is ideally placed to replicate biomolecular structures with tuneable building materials. Of particular interest are molecular nanopores, which transport cargo across membranes, as in DNA sequencing. Advanced nanopores control transport in response to triggers, but this cannot be easily replicated with biogenic proteins. Here we use DNA nanotechnology to build a synthetic molecular gate that opens in response to a specific protein. The gate self-assembles from six DNA strands to form a bilayer-spanning pore, and a lid strand comprising a protein-binding DNA aptamer to block the channel entrance. Addition of the trigger protein, thrombin, selectively opens the gate and enables a 330-fold increase inw the transport rate of small-molecule cargo. The molecular gate incorporates in delivery vesicles to controllably release enclosed cytotoxic drugs and kill eukaryotic cells. The generically designed gate may be applied in biomedicine, biosensing or for building synthetic cells.

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.

DNA Nanodevices with Selective Immune Cell Interaction and Function.

DNA nanotechnology produces precision nanostructures of defined chemistry. Expanding their use in biomedicine requires designed biomolecular interaction and function. Of topical interest are DNA nanostructures that function as vaccines with potential advantages over nonstructured nucleic acids in terms of serum stability and selective interaction with human immune cells. Here, we describe how compact DNA nanobarrels bind with a 400-fold selectivity via membrane anchors to white blood immune cells over erythrocytes, without affecting cell viability. The selectivity is based on the preference of the cholesterol lipid anchor for the more fluid immune cell membranes compared to the lower membrane fluidity of erythrocytes. Compacting DNA into the nanostructures gives rise to increased serum stability. The DNA barrels furthermore functionally modulate white blood cells by suppressing the immune response to pro-inflammatory endotoxin lipopolysaccharide. This is likely due to electrostatic or steric blocking of toll-like receptors on white blood cells. Our findings on immune cell-specific DNA nanostructures may be applied for vaccine development, immunomodulatory therapy to suppress septic shock, or the targeting of bioactive substances to immune cells.

A Biomimetic DNA-Based Membrane Gate for Protein-Controlled Transport of Cytotoxic Drugs.

Chemistry is ideally placed to replicate biomolecular structures with tuneable building materials. Of particular interest are molecular nanopores, which transport cargo across membranes, as in DNA sequencing. Advanced nanopores control transport in response to triggers, but this cannot be easily replicated with biogenic proteins. Here we use DNA nanotechnology to build a synthetic molecular gate that opens in response to a specific protein. The gate self-assembles from six DNA strands to form a bilayer-spanning pore, and a lid strand comprising a protein-binding DNA aptamer to block the channel entrance. Addition of the trigger protein, thrombin, selectively opens the gate and enables a 330-fold increase inw the transport rate of small-molecule cargo. The molecular gate incorporates in delivery vesicles to controllably release enclosed cytotoxic drugs and kill eukaryotic cells. The generically designed gate may be applied in biomedicine, biosensing or for building synthetic cells.

Design, assembly, and characterization of membrane-spanning DNA nanopores.

DNA nanopores are bio-inspired nanostructures that control molecular transport across lipid bilayer membranes. Researchers can readily engineer the structure and function of DNA nanopores to synergistically combine the strengths of DNA nanotechnology and nanopores. The pores can be harnessed in a wide range of areas, including biosensing, single-molecule chemistry, and single-molecule biophysics, as well as in cell biology and synthetic biology. Here, we provide a protocol for the rational design of nanobarrel-like DNA pores and larger DNA origami nanopores for targeted applications. We discuss strategies for the pores' chemical modification with lipid anchors to enable them to be inserted into membranes such as small unilamellar vesicles (SUVs) and planar lipid bilayers. The procedure covers the self-assembly of DNA nanopores via thermal annealing, their characterization using gel electrophoresis, purification, and direct visualization with transmission electron microscopy and atomic force microscopy. We also describe a gel assay to determine pore-membrane binding and discuss how to use single-channel current recordings and dye flux assays to confirm transport through the pores. We expect this protocol to take approximately 1 week to complete for DNA nanobarrel pores and 2-3 weeks for DNA origami pores.