Andrew J. Heron

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 α-hemolysin (αHL), are advantageous because they can be precisely manipulated by genetic modification. Here, we show that the transmembrane β-barrel of an engineered αHL 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.

Droplet interface bilayers

Droplet interface bilayers (DIBs) provide a superior platform for the biophysical analysis of membrane proteins. The versatile DIBs can also form networks, with features that include built-in batteries and sensors.

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.

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.

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.

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.

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.

Multiple Base‐Recognition Sites in a Biological Nanopore: Two Heads are Better than One

Ultra-rapid sequencing of DNA strands with nanopores is under intense investigation. The αHL protein nanopore is a leading candidate sensor for this approach. Multiple base-recognition sites have been identified in engineered αHL pores. By using immobilized synthetic oligonucleotides, we show here that additional sequence information can be gained when two recognition sites, rather than one, are employed within a single nanopore.

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.

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.