Stefan Howorka

Sequence-specific detection of individual DNA strands using engineered nanopores

We describe biosensor elements that are capable of identifying individual DNA strands with single-base resolution. Each biosensor element consists of an individual DNA oligonucleotide covalently attached within the lumen of the α-hemolysin (αHL) pore to form a “DNA–nanopore”. The binding of single-stranded DNA (ssDNA) molecules to the tethered DNA strand causes changes in the ionic current flowing through a nanopore. On the basis of DNA duplex lifetimes, the DNA–nanopores are able to discriminate between individual DNA strands up to 30 nucleotides in length differing by a single base substitution. This was exemplified by the detection of a drug resistance–conferring mutation in the reverse transcriptase gene of HIV. In addition, the approach was used to sequence a complete codon in an individual DNA strand tethered to a nanopore.

Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore.

Here we describe a new type of biosensor element for detecting proteins in solution at nanomolar concentrations. We tethered a 3.4 kDa polyethylene glycol chain at a defined site within the lumen of the transmembrane protein pore formed by staphylococcal α-hemolysin. The free end of the polymer was covalently attached to a biotin molecule. On incorporation of the modified pore into a lipid bilayer, the biotinyl group moves from one side of the membrane to the other, and is detected by reversible capture with a mutant streptavidin. The capture events are observed as changes in ionic current passing through single pores in planar bilayers. Accordingly, the modified pore allows detection of a protein analyte at the single-molecule level, facilitating both quantification and identification through a distinctive current signature. The approach has higher time resolution compared with other kinetic measurements, such as those obtained by surface plasmon resonance.

Kinetics of duplex formation for individual DNA strands within a single protein nanopore

A single oligonucleotide was covalently attached to a genetically engineered subunit of the heptameric protein pore, α-hemolysin, to allow DNA duplex formation inside the pore lumen. Single-channel current recording was used to study the properties of the modified pore. On addition of an oligonucleotide 8 bases in length and with a sequence complementary to the tethered DNA strand, current blockades with durations of hundreds of milliseconds occurred, representing hybridization events of individual oligonucleotides to the tethered DNA strand. Kinetic constants for DNA duplex formation at the single molecule level were derived and found to be consistent with established literature values for macroscopic duplex formation. The resultant equilibrium constant for duplex formation in the nanopore was found to be close to the experimentally derived constant for duplex formation in solution. A good agreement between the equilibrium constants for duplex formation in the nanopore and in solution was also found for two other oligonucleotide pairs. In addition, the nanopore recordings revealed details of the kinetics difficult to obtain by conventional methods, like surface plasmon resonance, which measure ensemble properties. By investigating the temperature dependence of DNA duplex formation at the single molecule level, the standard enthalpy and entropy of the interaction could be obtained.

Lipid-bilayer-spanning DNA nanopores with a bifunctional porphyrin anchor

Holding tight: An artificial membrane nanopore assembled from DNA oligonucleotides carries porphyrin tags (red), which anchor the nanostructure into the lipid bilayer. The porphyrin moieties also act as fluorescent dyes to aid the microscopic visualization of the DNA nanopore.

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 Ca2+ ion coordination. A Ca2+-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.

DNA-modified polymer pores allow pH- and voltage-gated control of channel flux.

Biological channels embedded in cell membranes regulate ionic transport by responding to external stimuli such as pH, voltage, and molecular binding. Mimicking the gating properties of these biological structures would be instrumental in the preparation of smart membranes used in biosensing, drug delivery, and ionic circuit construction. Here we present a new concept for building synthetic nanopores that can simultaneously respond to pH and transmembrane potential changes. DNA oligomers containing protonatable A and C bases are attached at the narrow opening of an asymmetric nanopore. Lowering the pH to 5.5 causes the positively charged DNA molecules to bind to other strands with negative backbones, thereby creating an electrostatic mesh that closes the pore to unprecedentedly high resistances of several tens of gigaohms. At neutral pH values, voltage switching causes the isolated DNA strands to undergo nanomechanical movement, as seen by a reversible current modulation. We provide evidence that the pH-dependent reversible closing mechanism is robust and applicable for nanopores with opening diameters of up to 14 nm. The concept of creating an electrostatic mesh may also be applied to different organic polymers.

A biomimetic DNA-based channel for the ligand-controlled transport of charged molecular cargo across a biological membrane

Biological ion channels are molecular gatekeepers that control transport across cell membranes. Recreating the functional principle of such systems and extending it beyond physiological ionic cargo is both scientifically exciting and technologically relevant to sensing or drug release. However, fabricating synthetic channels with a predictable structure remains a significant challenge. Here, we use DNA as a building material to create an atomistically determined molecular valve that can control when and which cargo is transported across a bilayer. The valve, which is made from seven concatenated DNA strands, can bind a specific ligand and, in response, undergo a nanomechanical change to open up the membrane-spanning channel. It is also able to distinguish with high selectivity the transport of small organic molecules that differ by the presence of a positively or negatively charged group. The DNA device could be used for controlled drug release and the building of synthetic cell-like or logic ionic networks.

Imaging Surface Charges of Individual Biomolecules

Surface charges play a key role in determining the structure and function of proteins, DNA, and larger biomolecular structures. Here we report on the measurement of the electrostatic surface potential of individual DNA and avidin molecules with nanometer resolution using Kelvin probe force microscopy. We also show, for the first time, the surface potential of buffer salts shielding individual DNA molecules, which would not be possible with conventional ensemble techniques.

A DNA Nanostructure for the Functional Assembly of Chemical Groups with Tunable Stoichiometry and Defined Nanoscale Geometry

Herein we describe a new approach which merges DNA-based nanostructures and the chemical modification of DNA.We show that tetrahedron-shaped nanostructures can act asscaffoldstoassembleamultitudeofdifferentchemicalgroupsat tunable stoichiometry and at geometrically defined sites.The resulting molecular entities exhibit functional propertiesbeneficial in biosensing and diagnostics. Our new strategy forassembling chemical groups at the nanoscale may beexpanded to endow other DNA structures with rationallydesigned functions.DNA tetrahedra, as intoduced by Goodman et al, arenanostructures with edges composed of double-strandedDNA (dsDNA; Figure 1a).

Rationally engineering natural protein assemblies in nanobiotechnology

Multimeric protein assemblies are essential components in viruses, bacteria, eukaryotic cells, and organisms where they act as cytoskeletal scaffold, storage containers, or for directional transport. The bottom-up structures can be exploited in nanobiotechnology by harnessing their built-in properties and combining them with new functional modules. This review summarizes the design principles of natural protein assemblies, highlights recent progress in their structural elucidation, and shows how rational engineering can create new biomaterials for applications in vaccine development, biocatalysis, materials science, and synthetic biology.