Lipidated DNA Nanostructures Target and Rupture Bacterial Membranes.

Chemistry has the power to endow supramolecular nanostructures with new biomedically relevant functions. Here it is reported that DNA nanostructures modified with cholesterol tags disrupt bacterial membranes to cause microbial cell death. The lipidated DNA nanostructures bind more readily to cholesterol-free bacterial membranes than to cholesterol-rich, eukaryotic membranes. These highly negatively charged, lipidated DNA nanostructures cause bacterial cell death by rupturing membranes. Strikingly, killing is mediated by clusters of barrel-shaped nanostructures that adhere to the membrane without the involvement of expected bilayer-puncturing barrels. These DNA nanomaterials may inspire the development of polymeric or small-molecule antibacterial agents that mimic the principles of selective binding and rupturing to help combat antimicrobial resistance.

Nanopores: synergy from DNA sequencing to industrial filtration - small holes with big impact.

Nanopores in thin membranes play important roles in science and industry. Single nanopores have provided a step-change in portable DNA sequencing and understanding nanoscale transport while multipore membranes facilitate food processing and purification of water and medicine. Despite the unifying use of nanopores, the fields of single nanopores and multipore membranes differ - to varying degrees - in terms of materials, fabrication, analysis, and applications. Such a partial disconnect hinders scientific progress as important challenges are best resolved together. This Viewpoint suggests how synergistic crosstalk between the two fields can provide considerable mutual benefits in fundamental understanding and the development of advanced membranes. We first describe the main differences including the atomistic definition of single pores compared to the less defined conduits in multipore membranes. We then outline steps to improve communication between the two fields such as harmonizing measurements and modelling of transport and selectivity. The resulting insight is expected to improve the rational design of porous membranes. The Viewpoint concludes with an outlook of other developments that can be best achieved by collaboration across the two fields to advance the understanding of transport in nanopores and create next-generation porous membranes tailored for sensing, filtration, and other applications.

Molecular Recognition in Confined Space Elucidated with DNA Nanopores and Single-Molecule Force Microscopy.

The binding of ligands to receptors within a nanoscale small space is relevant in biology, biosensing, and affinity filtration. Binding in confinement can be studied with biological systems but under the limitation that essential parameters cannot be easily controlled including receptor type and position within the confinement and its dimensions. Here we study molecular recognition with a synthetic confined nanopore with controllable pore dimension and molecular DNA receptors at different depth positions within the channel. Binding of a complementary DNA strand is studied at the single-molecule level with atomic force microscopy. Following the analysis, kinetic association rates are lower for receptors positioned deeper inside the pore lumen while dissociation is faster and requires less force. The phenomena are explained by the steric constraints on molecular interactions in confinement. Our study is the first to explore recognition in DNA nanostructures with atomic force microscopy and lays out new tools to further quantify the effect of nanoconfinement on molecular interactions.

Functional Nanopores Enabled with DNA.

Membrane-spanning nanopores are used in label-free single-molecule sensing and next-generation portable nucleic acid sequencing, and as powerful research tools in biology, biophysics, and synthetic biology. Naturally occurring protein and peptide pores, as well as synthetic inorganic nanopores, are used in these applications, with their limitations. The structural and functional repertoire of nanopores can be considerably expanded by functionalising existing pores with DNA strands and by creating an entirely new class of nanopores with DNA nanotechnology. This review outlines progress in this area of functional DNA nanopores and outlines developments to open up new 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.

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.

Hydrophobic Interactions between DNA Duplexes and Synthetic and Biological Membranes.

Equipping DNA with hydrophobic anchors enables targeted interaction with lipid bilayers for applications in biophysics, cell biology, and synthetic biology. Understanding DNA-membrane interactions is crucial for rationally designing functional DNA. Here we study the interactions of hydrophobically tagged DNA with synthetic and cell membranes using a combination of experiments and atomistic molecular dynamics (MD) simulations. The DNA duplexes are rendered hydrophobic by conjugation to a terminal cholesterol anchor or by chemical synthesis of a charge-neutralized alkyl-phosphorothioate (PPT) belt. Cholesterol-DNA tethers to lipid vesicles of different lipid compositions and charges, while PPT DNA binding strongly depends on alkyl length, belt position, and headgroup charge. Divalent cations in the buffer can also influence binding. Our MD simulations directly reveal the complex structure and energetics of PPT DNA within a lipid membrane, demonstrating that longer alkyl-PPT chains provide the most stable membrane anchoring but may disrupt DNA base paring in solution. When tested on cells, cholesterol-DNA is homogeneously distributed on the cell surface, while alkyl-PPT DNA accumulates in clustered structures on the plasma membrane. DNA tethered to the outside of the cell membrane is distinguished from DNA spanning the membrane by nuclease and sphingomyelinase digestion assays. The gained fundamental insight on DNA-bilayer interactions will guide the rational design of membrane-targeting nanostructures.

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.

Exploring the Relationship between BODIPY Structure and Spectroscopic Properties to Design Fluorophores for Bioimaging.

Designing chromophores for biological applications requires a fundamental understanding of how the chemical structure of a chromophore influences its photophysical properties. We here describe the synthesis of a library of BODIPY dyes, exploring diversity at various positions around the BODIPY core. The results show that the nature and position of substituents have a dramatic effect on the spectroscopic properties. Substituting in a heavy atom or adjusting the size and orientation of a conjugated system provides a means of altering the spectroscopic profiles with high precision. The insight from the structure-activity relationship was applied to devise a new BODIPY dye with rationally designed photochemical properties including absorption towards the near-infrared region. The dye also exhibited switch-on fluorescence to enable visualisation of cells with high signal-to-noise ratio without washing-out of unbound dye. The BODIPY-based probe is non-cytotoxic and compatible with staining procedures including cell fixation and immunofluorescence microscopy.

Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG.

Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α and γ classes). They provide a fitness advantage in pathogenic strains and induce a strong pro-inflammatory response during bacteraemia. Curli formation requires a dedicated protein secretion machinery comprising the outer membrane lipoprotein CsgG and two soluble accessory proteins, CsgE and CsgF. Here we report the X-ray structure of Escherichia coli CsgG in a non-lipidated, soluble form as well as in its native membrane-extracted conformation. CsgG forms an oligomeric transport complex composed of nine anticodon-binding-domain-like units that give rise to a 36-stranded ß-barrel that traverses the bilayer and is connected to a cage-like vestibule in the periplasm. The transmembrane and periplasmic domains are separated by a 0.9-nm channel constriction composed of three stacked concentric phenylalanine, asparagine and tyrosine rings that may guide the extended polypeptide substrate through the secretion pore. The specificity factor CsgE forms a nonameric adaptor that binds and closes off the periplasmic face of the secretion channel, creating a 24,000 Å(3) pre-constriction chamber. Our structural, functional and electrophysiological analyses imply that CsgG is an ungated, non-selective protein secretion channel that is expected to employ a diffusion-based, entropy-driven transport mechanism.

Spatial Presentation of Cholesterol Units on a DNA Cube as a Determinant of Membrane Protein-Mimicking Functions.

Cells use membrane proteins as gatekeepers to transport ions and molecules, catalyze reactions, relay signals, and interact with other cells. DNA nanostructures with lipidic anchors are promising as membrane protein mimics because of their high tunability. However, the design features specifying DNA nanostructures' functions in lipid membranes are yet to be fully understood. Here, we show that altering patterns of cholesterol units on a cubic DNA scaffold dramatically changes its interaction mode with lipid membranes. This results in simple design rules that allow a single DNA nanostructure to reproduce multiple membrane protein functions: peripheral anchoring, nanopore behavior, and conformational switching to reveal membrane-binding units. Strikingly, the DNA-cholesterol cubes constitute the first open-walled DNA nanopores, as only a quarter of their wall is made of DNA. This functional diversity can increase our fundamental understanding of membrane phenomena and result in sensing, drug delivery, and cell manipulation tools.

Cholesterol Anchors Enable Efficient Binding and Intracellular Uptake of DNA Nanostructures.

DNA nanostructures constitute a rapidly advancing tool-set for exploring cell-membrane functions and intracellular sensing or advancing delivery of biomolecular cargo  into cells. Chemical conjugation with lipid anchors can mediate binding of DNA nanostructures to synthetic lipid bilayers, yet how such structures interact with biological membranes and internalize cells has not been shown. Here, an archetypal 6-duplex nanobundle is used to investigate how lipid conjugation influences DNA cell binding and internalization kinetics. Cellular interactions of DNA nanobundles modified with one and three cholesterol anchors were assessed using flow cytometry and confocal microscopy. Nuclease digestion was used to distinguish surface-bound DNA, which is nuclease accessible, from internalized DNA. Three cholesterol anchors were found to enhance cellular association by up to 10-fold when compared with unmodified DNA. The bundles were endocytosed efficiently within 24 h. The results can help design controlled DNA binding and trafficking into cells.

Solvent-dependent photophysics of a red-shifted, biocompatible coumarin photocage.

Controlling the activity of biomolecules with light-triggered photocages is an important research tool in the life sciences. We describe here a coumarin photocage that unusually combines the biocompatible optical properties of strong absorption at a long wavelength close to 500 nm and high photolysis quantum yields. The favourable properties are achieved by synthetically installing on the photocage scaffold a diethyl amino styryl moiety and a thionoester group rather than the lactone typical for coumarins. The photocage's photophysics are analysed with microsecond transient absorption spectroscopy to reveal the nature of the excited state in the photolysis pathway. The excited state is found to be strongly dependent on solvent polarity with a triplet state formed in DMSO and a charge-separated state in water that is likely due to aggregation. A long triplet lifetime is also correlated with a high photolysis quantum yield. Our study on the biocompatible photocage reveals fundamental insight for designing advanced photocages such as longer wavelengths in different solvent conditions tailored for applications in basic and applied research.

A Photo-responsive Small-Molecule Approach for the Opto-epigenetic Modulation of DNA Methylation.

Controlling the functional dynamics of DNA within living cells is essential in biomedical research. Epigenetic modifications such as DNA methylation play a key role in this endeavour. DNA methylation can be controlled by genetic means. Yet there are few chemical tools available for the spatial and temporal modulation of this modification. Herein, we present a small-molecule approach to modulate DNA methylation with light. The strategy uses a photo-tuneable version of a clinically used drug (5-aza-2'-deoxycytidine) to alter the catalytic activity of DNA methyltransferases, the enzymes that methylate DNA. After uptake by cells, the photo-regulated molecule can be light-controlled to reduce genome-wide DNA methylation levels in proliferating cells. The chemical tool complements genetic, biochemical, and pharmacological approaches to study the role of DNA methylation in biology and medicine.

Dynamic Interactions between Lipid-Tethered DNA and Phospholipid Membranes.

Lipid-anchored DNA can attach functional cargo to bilayer membranes in DNA nanotechnology, synthetic biology, and cell biology research. To optimize DNA anchoring, an understanding of DNA-membrane interactions in terms of binding strength, extent, and structural dynamics is required. Here we use experiments and molecular dynamics (MD) simulations to determine how the membrane binding of cholesterol-modified DNA depends on electrostatic and steric factors involving the lipid headgroup charge, duplexed or single-stranded DNA, and the buffer composition. The experiments distinguish between free and membrane vesicle-bound DNA and thereby reveal the surface density of anchored DNA and its binding affinity, something which had previously not been known. The Kd values range from 8.5 ± 4.9 to 466 ± 134 µM whereby negatively charged headgroups led to weak binding due to the electrostatic repulsion with respect to the negatively charged DNA. Atomistic MD simulations explain the findings and elucidate the dynamic nature of anchored DNA such as the mushroom-like conformation of single-stranded DNA hovering over the bilayer surface in contrast to a straight-up conformation of double-stranded DNA. The biophysical insight into the binding strength to membranes as well as the molecular accessibility of DNA for hybridization to molecular cargo is expected to facilitate the creation of biomimetic DNA versions of natural membrane nanopores and cytoskeletons for research and nanobiotechnology.

Tunable DNA Hybridization Enables Spatially and Temporally Controlled Surface-Anchoring of Biomolecular Cargo.

The controlled immobilization of biomolecules onto surfaces is relevant in biosensing and cell biological research. Spatial control is achieved by surface-tethering molecules in micro- or nanoscale patterns. Yet, there is an increasing demand for temporal control over how long biomolecular cargo stays immobilized until released into the medium. Here, we present a DNA hybridization-based approach to reversibly anchor biomolecular cargo onto micropatterned surfaces. Cargo is linked to a DNA oligonucleotide that hybridizes to a sequence-complementary, surface-tethered strand. The cargo is released from the substrate by the addition of an oligonucleotide that disrupts the duplex interaction via toehold-mediated strand displacement. The unbound tether strand can be reloaded. The generic strategy is implemented with small-molecule or protein cargo, varying DNA sequences, and multiple surface patterning routes. The approach may be used as a tool in biological research to switch membrane proteins from a locally fixed to a free state, or in biosensing to shed biomolecular receptors to regenerate the sensor surface.

SbsB structure and lattice reconstruction unveil Ca2+ triggered S-layer assembly.

S-layers are regular two-dimensional semipermeable protein layers that constitute a major cell-wall component in archaea and many bacteria. The nanoscale repeat structure of the S-layer lattices and their self-assembly from S-layer proteins (SLPs) have sparked interest in their use as patterning and display scaffolds for a range of nano-biotechnological applications. Despite their biological abundance and the technological interest in them, structural information about SLPs is limited to truncated and assembly-negative proteins. Here we report the X-ray structure of the SbsB SLP of Geobacillus stearothermophilus PV72/p2 by the use of nanobody-aided crystallization. SbsB consists of a seven-domain protein, formed by an amino-terminal cell-wall attachment domain and six consecutive immunoglobulin-like domains, that organize into a φ-shaped disk-like monomeric crystallization unit stabilized by interdomain Ca(2+) ion coordination. A Ca(2+)-dependent switch to the condensed SbsB quaternary structure pre-positions intermolecular contact zones and renders the protein competent for S-layer assembly. On the basis of crystal packing, chemical crosslinking data and cryo-electron microscopy projections, we present a model for the molecular organization of this SLP into a porous protein sheet inside the S-layer. The SbsB lattice represents a previously undescribed structural model for protein assemblies and may advance our understanding of SLP physiology and self-assembly, as well as the rational design of engineered higher-order structures for biotechnology.

Co-Immobilization of Proteins and DNA Origami Nanoplates to Produce High-Contrast Biomolecular Nanoarrays.

The biofunctionalization of nanopatterned surfaces with DNA origami nanostructures is an important topic in nanobiotechnology. An unexplored challenge is, however, to co-immobilize proteins with DNA origami at pre-determined substrate sites in high contrast relative to the nontarget areas. The immobilization should, in addition, preferably be achieved on a transparent substrate to allow ultrasensitive optical detection. If successful, specific co-binding would be a step towards stoichiometrically defined arrays with few to individual protein molecules per site. Here, we successfully immobilize with high specificity positively charged avidin proteins and negatively charged DNA origami nanoplates on 100 nm-wide carbon nanoislands while suppressing undesired adsorption to surrounding nontarget areas. The arrays on glass slides achieve unprecedented selectivity factors of up to 4000 and allow ultrasensitive fluorescence read-out. The co-immobilization onto the nanoislands leads to layered biomolecular architectures, which are functional because bound DNA origami influences the number of capturing sites on the nanopatches for other proteins. The novel hybrid DNA origami-protein nanoarrays allow the fabrication of versatile research platforms for applications in biosensing, biophysics, and cell biology, and, in addition, represent an important step towards single-molecule protein arrays.

Dendrimers in Nanoscale Confinement: The Interplay between Conformational Change and Nanopore Entrance.

Hyperbranched dendrimers are nanocarriers for drugs, imaging agents, and catalysts. Their nanoscale confinement is of fundamental interest and occurs when dendrimers with bioactive payload block or pass biological nanochannels or when catalysts are entrapped in inorganic nanoporous support scaffolds. The molecular process of confinement and its effect on dendrimer conformations are, however, poorly understood. Here, we use single-molecule nanopore measurements and molecular dynamics simulations to establish an atomically detailed model of pore dendrimer interactions. We discover and explain that electrophoretic migration of polycationic PAMAM dendrimers into confined space is not dictated by the diameter of the branched molecules but by their size and generation-dependent compressibility. Differences in structural flexibility also rationalize the apparent anomaly that the experimental nanopore current read-out depends in nonlinear fashion on dendrimer size. Nanoscale confinement is inferred to reduce the protonation of the polycationic structures. Our model can likely be expanded to other dendrimers and be applied to improve the analysis of biophysical experiments, rationally design functional materials such as nanoporous filtration devices or nanoscale drug carriers that effectively pass biological pores.

Biomimetic Hybrid Nanocontainers with Selective Permeability.

Chemistry plays a crucial role in creating synthetic analogues of biomacromolecular structures. Of particular scientific and technological interest are biomimetic vesicles that are inspired by natural membrane compartments and organelles but avoid their drawbacks, such as membrane instability and limited control over cargo transport across the boundaries. In this study, completely synthetic vesicles were developed from stable polymeric walls and easy-to-engineer membrane DNA nanopores. The hybrid nanocontainers feature selective permeability and permit the transport of organic molecules of 1.5 nm size. Larger enzymes (ca. 5 nm) can be encapsulated and retained within the vesicles yet remain catalytically active. The hybrid structures constitute a new type of enzymatic nanoreactor. The high tunability of the polymeric vesicles and DNA pores will be key in tailoring the nanocontainers for applications in drug delivery, bioimaging, biocatalysis, and cell mimicry.