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 Light-Triggered Synthetic Nanopore for Controlling Molecular Transport Across Biological Membranes.

Controlling biological molecular processes with light is of interest in biological research and biomedicine, as light allows precise and selective activation in a non-invasive and non-toxic manner. A molecular process benefitting from light control is the transport of cargo across biological membranes, which is conventionally achieved by membrane-puncturing barrel-shaped nanopores. Yet, there is also considerable gain in constructing more complex gated pores. Here, we pioneer a synthetic light-gated nanostructure which regulates transport across membranes via a controllable lid. The light-triggered nanopore is self-assembled from six pore-forming DNA strands and a lid strand carrying light-switchable azobenzene molecules. Exposure to light opens the pore to allow small-molecule transport across membranes. Our light-triggered pore advances biomimetic chemistry and DNA nanotechnology and may be used in biotechnology, biosensing, targeted drug release, or synthetic cells.

Nanopore DNA sequencing technologies and their applications towards single-molecule proteomics.

Sequencing of nucleic acids with nanopores has emerged as a powerful tool offering rapid readout, high accuracy, low cost and portability. This label-free method for sequencing at the single-molecule level is an achievement on its own. However, nanopores also show promise for the technologically even more challenging sequencing of polypeptides, something that could considerably benefit biological discovery, clinical diagnostics and homeland security, as current techniques lack portability and speed. Here we survey the biochemical innovations underpinning commercial and academic nanopore DNA/RNA sequencing techniques, and explore how these advances can fuel developments in future protein sequencing with nanopores.

Multi-Stimuli-Responsive and Mechano-Actuated Biomimetic Membrane Nanopores Self-Assembled from DNA.

In bioinspired design, biological templates are mimicked in structure and function by highly controllable synthetic means. Of interest are static barrel-like nanopores that enable molecular transport across membranes for use in biosensing, sequencing, and biotechnology. However, biological ion channels offer additional functions such as dynamic changes of the entire pore shape between open and closed states, and triggering of dynamic processes with biochemical and physical stimuli. To better capture this complexity, this report presents multi-stimuli and mechano-responsive biomimetic nanopores which are created with DNA nanotechnology. The nanopores switch between open and closed states, whereby specific binding of DNA and protein molecules as stimuli locks the pores in the open state. Furthermore, the physical stimulus of high transmembrane voltage switches the pores into a closed state. In addition, the pore diameters are larger and more tunable than those of natural templates. These multi-stimuli-responsive and mechanically actuated nanopores mimic several aspects of complex biological channels yet offer easier control over pore size, shape and stimulus response. The designer pores are expected to be applied in biosensing and synthetic biology.

Highly shape- and size-tunable membrane nanopores made with DNA.

Membrane nanopores are key for molecular transport in biology, portable DNA sequencing1-4, label-free single-molecule analysis5-14 and nanomedicine5. Transport traditionally relies on barrel-like channels of a few nanometres width, but there is considerable scientific and technological interest for much wider structures of tunable shape. Yet, these nanopores do not exist in nature and are challenging to build using existing de novo routes for proteins10,15-17. Here, we show that rational design with DNA can drastically expand the structural and functional range of membrane nanopores. Our design strategy bundles DNA duplexes into pore subunits that modularly arrange to form tunable pore shapes and lumen widths of up to tens of nanometres. Functional units for recognition or signalling can be optionally attached. By dialling in essential parameters, we demonstrate the utility and potential of the custom-engineered nanopores by electrical direct single-molecule sensing of 10-nm-sized proteins using widely used research and hand-held analysis devices. The designer nanopores illustrate how DNA nanotechnology can deliver functional biomolecular structures to be used in synthetic biology, single-molecule enzymology and biophysical analysis, as well as portable diagnostics and environmental screening.

A reversibly gated protein-transporting membrane channel made of DNA.

Controlled transport of biomolecules across lipid bilayer membranes is of profound significance in biological processes. In cells, cargo exchange is mediated by dedicated channels that respond to triggers, undergo a nanomechanical change to reversibly open, and thus regulate cargo flux. Replicating these processes with simple yet programmable chemical means is of fundamental scientific interest. Artificial systems that go beyond nature's remit in transport control and cargo are also of considerable interest for biotechnological applications but challenging to build. Here, we describe a synthetic channel that allows precisely timed, stimulus-controlled transport of folded and functional proteins across bilayer membranes. The channel is made via DNA nanotechnology design principles and features a 416 nm2 opening cross-section and a nanomechanical lid which can be controllably closed and re-opened via a lock-and-key mechanism. We envision that the functional DNA device may be used in highly sensitive biosensing, drug delivery of proteins, and the creation of artificial cell networks.

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.

Synthetic protein-conductive membrane nanopores built with DNA.

Nanopores are key in portable sequencing and research given their ability to transport elongated DNA or small bioactive molecules through narrow transmembrane channels. Transport of folded proteins could lead to similar scientific and technological benefits. Yet this has not been realised due to the shortage of wide and structurally defined natural pores. Here we report that a synthetic nanopore designed via DNA nanotechnology can accommodate folded proteins. Transport of fluorescent proteins through single pores is kinetically analysed using massively parallel optical readout with transparent silicon-on-insulator cavity chips vs. electrical recordings to reveal an at least 20-fold higher speed for the electrically driven movement. Pores nevertheless allow a high diffusive flux of more than 66 molecules per second that can also be directed beyond equillibria. The pores may be exploited to sense diagnostically relevant proteins with portable analysis technology, to create molecular gates for drug delivery, or to build synthetic cells.