Highly parallel direct RNA sequencing on an array of nanopores.

Sequencing the RNA in a biological sample can unlock a wealth of information, including the identity of bacteria and viruses, the nuances of alternative splicing or the transcriptional state of organisms. However, current methods have limitations due to short read lengths and reverse transcription or amplification biases. Here we demonstrate nanopore direct RNA-seq, a highly parallel, real-time, single-molecule method that circumvents reverse transcription or amplification steps. This method yields full-length, strand-specific RNA sequences and enables the direct detection of nucleotide analogs in RNA.

Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore.

The sequencing of individual DNA strands with nanopores is under investigation as a rapid, low-cost platform in which bases are identified in order as the DNA strand is transported through a pore under an electrical potential. Although the preparation of solid-state nanopores is improving, biological nanopores, such as alpha-hemolysin (alphaHL), are advantageous because they can be precisely manipulated by genetic modification. Here, we show that the transmembrane beta-barrel of an engineered alphaHL pore contains 3 recognition sites that can be used to identify all 4 DNA bases in an immobilized single-stranded DNA molecule, whether they are located in an otherwise homopolymeric DNA strand or in a heteropolymeric strand. The additional steps required to enable nanopore DNA sequencing are outlined.

Fluorinated amphiphiles control the insertion of α-hemolysin pores into lipid bilayers.

The insertion of fully folded and assembled ion channels and pores into planar lipid bilayers for electrical recording has been facilitated by the use of conventional detergents at a final concentration below the critical micelle concentration (CMC). After the desired number of channels or pores (often one) has been incorporated into a bilayer, it is important to prevent further insertion events, which is often done by awkward techniques such as perfusion. Here, we show that the addition of single-chain fluorinated amphiphiles (F-amphiphiles) with zwitterionic, simple neutral, and neutral oligomeric headgroups at a concentration above the CMC prevents the further insertion of staphylococcal α-hemolysin pores, MspA pores, and Kcv potassium channels into lipid bilayers. We found the commercially available F(6)FC (fluorinated fos-choline with a C(6)F(13)C(2)H(4) chain) to be the least perturbing and most effective agent for this purpose. Bilayers are known to be resistant to F-amphiphiles, which in this case we suppose sequester the pores and channels within amphiphile aggregates. We suggest that F-amphiphiles might be useful in the fabrication of bilayer arrays for nanopore sensor devices and the rapid screening of membrane proteins.

Molecular dynamics simulations of DNA within a nanopore: arginine-phosphate tethering and a binding/sliding mechanism for translocation.

Protein nanopores show great potential as low-cost detectors in DNA sequencing devices. To date, research has largely focused on the staphylococcal pore α-hemolysin (αHL). In the present study, we have developed simplified models of the wild-type αHL pore and various mutants in order to study the translocation dynamics of single-stranded DNA under the influence of an applied electric field. The model nanopores reflect the experimentally measured conductance values in planar lipid bilayers. We show that interactions between rings of cationic amino acids and DNA backbone phosphates result in metastable tethering of nucleic acid molecules within the pore, leading us to propose a "binding and sliding" mechanism for translocation. We also observe folding of DNA into nonlinear conformational intermediates during passage through the confined nanopore environment. Despite adopting nonlinear conformations, the DNA hexamer always exits the pore in the same orientation as it enters (3' to 5') in our simulations. The observations from our simulations help to rationalize experimentally determined trends in residual current and translocation efficiency for αHL and its mutants.

Determining membrane capacitance by dynamic control of droplet interface bilayer area.

By making dynamic changes to the area of a droplet interface bilayer (DIB), we are able to measure the specific capacitance of lipid bilayers with improved accuracy and precision over existing methods. The dependence of membrane specific capacitance on the chain-length of the alkane oil present in the bilayer is similar to that observed in black lipid membranes. In contrast to conventional artificial bilayers, DIBs are not confined by an aperture, which enables us to determine that the dependence of whole bilayer capacitance on applied potential is predominantly a result of a spontaneous increase in bilayer area. This area change arises from the creation of new bilayer at the three phase interface and is driven by changes in surface tension with applied potential that can be described by the Young-Lippmann equation. By accounting for this area change, we are able to determine the proportion of the capacitance dependence that arises from a change in specific capacitance with applied potential. This method provides a new tool with which to investigate the vertical compression of the bilayer and understand the changes in bilayer thickness with applied potential. We find that, for 1,2-diphytanoyl-sn-glycero-3-phosphocholine membranes in hexadecane, specific bilayer capacitance varies by 0.6-1.5% over an applied potential of ±100 mV.

Nucleobase recognition in ssDNA at the central constriction of the alpha-hemolysin pore.

Nanopores are under investigation for single-molecule DNA sequencing. The alpha-hemolysin (alphaHL) protein nanopore contains three recognition points capable of nucleobase discrimination in individual immobilized ssDNA molecules. We have modified the recognition point R(1) by extensive mutagenesis of residue 113. Amino acids that provide an energy barrier to ion flow (e.g., bulky or hydrophobic residues) strengthen base identification, while amino acids that lower the barrier weaken it. Amino acids with related side chains produce similar patterns of nucleobase recognition providing a rationale for the redesign of recognition points.

Simultaneous measurement of ionic current and fluorescence from single protein pores.

The ability to simultaneously monitor both the ionic current and fluorescence from membrane channels and pores has the potential to link structural changes with function in such proteins. We present a new method for simultaneously measuring single-channel electrical currents and fluorescence from membrane proteins by using water-in-oil droplet bilayers. We demonstrate the simultaneous fluorescence and electrical detection of stochastic blocking by cyclodextrin in multiple staphylococcal alpha-hemolysin pores. The combined fluorescence signal from individual pores exhibits the same sequence of blocking events as the total current recording, showing that the two signals from each pore are correlated.

Direct detection of membrane channels from gels using water-in-oil droplet bilayers.

We form planar lipid bilayers between an aqueous droplet and a hydrogel support immersed in a lipid-oil solution. By scanning the bilayer over the surface of an SDS-PAGE gel, we are able to directly detect membrane proteins from gels using single-channel recording. Using this technique, we are able to examine low levels of endogenous protein from cell extracts without the need for over-expression. We also use droplet bilayers to detect small molecules from hydrogels. The bilayers show enhanced stability compared to conventional planar lipid bilayers, and both bilayer size and position can be controlled during an experiment. Hydrogel scanning with droplet bilayers provides a new method for the discovery and characterization of ion channels with the potential for high-throughput screening.

New technologies for DNA analysis--a review of the READNA Project.

The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 41/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.

Formation of droplet networks that function in aqueous environments.

Aqueous droplets in oil that are coated with lipid monolayers and joined through interface bilayers are useful for biophysical measurements on membrane proteins. Functional networks of droplets that can act as light sensors, batteries and electrical components can also be made by incorporating pumps, channels and pores into the bilayers. These networks of droplets mimic simple tissues, but so far have not been used in physiological environments because they have been constrained to a bulk oil phase. Here, we form structures called multisomes in which networks of aqueous droplets with defined compositions are encapsulated within small drops of oil in water. The encapsulated droplets adhere to one another and to the surface of the oil drop to form interface bilayers that allow them to communicate with each other and with the surrounding aqueous environment through membrane pores. The contents in the droplets can be released by changing the pH or temperature of the surrounding solution. The multicompartment framework of multisomes mimics a tissue and has potential applications in synthetic biology and medicine.

Analysis of single nucleic acid molecules with protein nanopores.

We describe the methods used in our laboratory for the analysis of single nucleic acid molecules with protein nanopores. The technical section is preceded by a review of the variety of experiments that can be done with protein nanopores. The end goal of much of this work is single-molecule DNA sequencing, although sequencing is not discussed explicitly here. The technical section covers the equipment required for nucleic acid analysis, the preparation and storage of the necessary materials, and aspects of signal processing and data analysis.

Identification of epigenetic DNA modifications with a protein nanopore.

Two DNA bases, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (hmC), marks of epigenetic modification, are recognized in immobilized DNA strands and distinguished from G, A, T and C by nanopore current recording. Therefore, if further aspects of nanopore sequencing can be addressed, the approach will provide a means to locate epigenetic modifications in unamplified genomic DNA.

A high pressure cell for simultaneous osmotic pressure and x-ray diffraction measurements.

In this paper, we report on a novel osmotic cell, developed to simultaneously subject a sample to osmotic stress and measure structural changes by small angle x-ray diffraction. The osmotic cell offers many advantages over more conventional methods of osmotically stressing soft materials to measure their structural response. In particular, a full osmotic analysis can be performed with a single small sample (25 microl). This reduces sample handling and the associated systematic errors, as well as enabling tight control and monitoring of the thermodynamic environment during osmosis, thereby increasing measurement precision. The cell design enables control of osmotic pressure to +/-0.04 bar over a pressure range of 1-100 bar, and temperature control to +/-0.05 degrees C. Under these conditions, the lattice spacing in lyotropic structures was resolved to better than +/-0.005 A. Using the osmotic cell, we demonstrate good agreement with previous conventional measurements on the energy of dehydrating the fluid lamellar phase of dioleoylphosphatidylcholine in water.

Droplet networks with incorporated protein diodes show collective properties.

Recently, we demonstrated that submicrolitre aqueous droplets submerged in an apolar liquid containing lipid can be tightly connected by means of lipid bilayers to form networks. Droplet interface bilayers have been used for rapid screening of membrane proteins and to form asymmetric bilayers with which to examine the fundamental properties of channels and pores. Networks, meanwhile, have been used to form microscale batteries and to detect light. Here, we develop an engineered protein pore with diode-like properties that can be incorporated into droplet interface bilayers in droplet networks to form devices with electrical properties including those of a current limiter, a half-wave rectifier and a full-wave rectifier. The droplet approach, which uses unsophisticated components (oil, lipid, salt water and a simple pore), can therefore be used to create multidroplet networks with collective properties that cannot be produced by droplet pairs.

Enhanced stability and fluidity in droplet on hydrogel bilayers for measuring membrane protein diffusion.

We form artificial lipid bilayers suitable for single-molecule fluorescence microscopy by contacting an aqueous droplet with a hydrogel support immersed in a solution of lipid in oil. Our results show that droplet on hydrogel bilayers (DHBs) have high lipid mobilities, similar to those observed in unsupported lipid bilayers. DHBs are also stable over a period of several weeks. We examine membrane protein diffusion in these bilayers and report a decreased lateral mobility of the heptameric beta-barrel pore-forming toxin alpha-hemolysin versus that of its monomeric precursor. These results corroborate previous models of the alpha-hemolysin insertion mechanism where the monomer binds to the lipid bilayer without insertion.

The diversity of the liquid ordered (Lo) phase of phosphatidylcholine/cholesterol membranes: a variable temperature multinuclear solid-state NMR and x-ray diffraction study.

To investigate the properties of a pure liquid ordered (Lo) phase in a model membrane system, a series of saturated phosphatidylcholines combined with cholesterol were examined by variable temperature multinuclear (1H, 2H, 13C, 31P) solid-state NMR spectroscopy and x-ray scattering. Compositions with cholesterol concentrations>or=40 mol %, well within the Lo phase region, are shown to exhibit changes in properties as a function of temperature and cholesterol content. The 2H-NMR data of both cholesterol and phospholipids were used to more accurately map the Lo phase boundary. It has been established that the gel-Lo phase coexistence extends to 60 mol % cholesterol and a modified phase diagram is presented. Combined 1H-, 2H-, 13C-NMR, and x-ray scattering data indicate that there are large changes within the Lo phase region, in particular, 1H-magic angle spinning NMR and wide-angle x-ray scattering were used to examine the in-plane intermolecular spacing, which approaches that of a fluid Lalpha phase at high temperature and high cholesterol concentrations. Although it is well known for cholesterol to broaden the gel-to-fluid transition temperature, we have observed, from the 13C magic angle spinning NMR data, that the glycerol region can still undergo a "melting", though this is broadened with increasing cholesterol content and changes with phospholipid chain length. Also from 2H-NMR order parameter data it was observed that the effect of temperature on chain length became smaller with increasing cholesterol content. Finally, from the cholesterol order parameter, it has been previously suggested that it is possible to determine the degree to which cholesterol associates with different phospholipids. However, we have found that by taking into account the relative temperature above the phase boundary this relationship may not be correct.

Pressure-jump X-ray studies of liquid crystal transitions in lipids.

In this paper, we give an overview of our studies by static and time-resolved X-ray diffraction of inverse cubic phases and phase transitions in lipids. In [section sign] 1, we briefly discuss the lyotropic phase behaviour of lipids, focusing attention on non-lamellar structures, and their geometric/topological relationship to fusion processes in lipid membranes. Possible pathways for transitions between different cubic phases are also outlined. In [section sign] 2, we discuss the effects of hydrostatic pressure on lipid membranes and lipid phase transitions, and describe how the parameters required to predict the pressure dependence of lipid phase transition temperatures can be conveniently measured. We review some earlier results of inverse bicontinuous cubic phases from our laboratory, showing effects such as pressure-induced formation and swelling. In [section sign] 3, we describe the technique of pressure-jump synchrotron X-ray diffraction. We present results that have been obtained from the lipid system 1:2 dilauroylphosphatidylcholine/lauric acid for cubic-inverse hexagonal, cubic-cubic and lamellar-cubic transitions. The rate of transition was found to increase with the amplitude of the pressure-jump and with increasing temperature. Evidence for intermediate structures occurring transiently during the transitions was also obtained. In [section sign] 4, we describe an IDL-based 'AXcess' software package being developed in our laboratory to permit batch processing and analysis of the large X-ray datasets produced by pressure-jump synchrotron experiments. In [section sign] 5, we present some recent results on the fluid lamellar-Pn3m cubic phase transition of the single-chain lipid 1-monoelaidin, which we have studied both by pressure-jump and temperature-jump X-ray diffraction. Finally, in [section sign] 6, we give a few indicators of future directions of this research. We anticipate that the most useful technical advance will be the development of pressure-jump apparatus on the microsecond time-scale, which will involve the use of a stack of piezoelectric pressure actuators. The pressure-jump technique is not restricted to lipid phase transitions, but can be used to study a wide range of soft matter transitions, ranging from protein unfolding and DNA unwinding and transitions, to phase transitions in thermotropic liquid crystals, surfactants and block copolymers.