Hybrid Nanopores for Single-Molecule Sensing

Lead Research Organisation: University of Oxford
Department Name: Oxford Chemistry


Stochastic sensing with nanopores is a versatile technology that can be used for the recognition and quantification of a wide range of substances (known as analytes) through the detection of individual molecules. Our partner company, Oxford Nanopore Technologies, has been incorporating stochastic sensing into next-generation hand-held devices. The most highly developed application at Oxford Nanopore is cheap, extremely rapid DNA sequencing, which promises to revolutionise numerous areas of biology including aspects of medicine, ancestry and forensics. Currently, a portable sequencer is being tested at hundreds of sites worldwide.

In stochastic sensing, analytes are detected as they enter and leave a single narrow pore perturbing a current that flows through it. The diameters of the pores, known as nanopores, are similar to those of a small molecule, about one-fifty thousandth of the diameter of a human hair, providing the basis for detection by current perturbation. Typically current changes of the order of one trillionth of an ampere are measured. Analytes have included drug molecules and small molecules found in the body that act as markers for disease. In the case of DNA sequencing, individual bases are detected as an extended DNA strand is threaded through a nanopore.

Protein pores are advantageous for stochastic sensing, because they can be modified for particular applications with atomic precision and prepared in near homogeneous form. Until now, very narrow protein pores have been used and therefore stochastic sensing has been limited to analytes of small size or, in the case of DNA, to extended polymer chains. In the proposed work, we will endeavour to make a new class of functional nanopores, DNA-Protein hybrid nanopores. These pores will be constructed from folded DNA, known as DNA origami, and protein components. The DNA will act as a scaffold for the protein, ensuring that the new pores are up to fifteen times larger in internal diameter than the pores used before. Further, each pore will be of identical size and no incompletes pores will be present, a goal that has not be achieved previously. Finally, it will be possible to modify the new pores at precisely determined sites, which cannot be done with competing technologies, such as solid-state pores.

The DNA-protein hybrid nanopores will enable a critical step forward for stochastic sensing by allowing the detection of a wide range of large biological molecules that can enter the pores, including proteins, DNAs and polymeric sugars. Conversely, it will also be possible to lodge these large molecules within the hybrid pores, where they will act as binding sites for a variety of additional analytes. In a futuristic application, it may prove possible to sequence double-stranded DNAs with hybrid pores, which will provide a significant advantage over the manipulations currently required for nanopore sequencing.

Our industrial partner, Oxford Nanopore, will evaluate and test our most promising DNA-protein hybrid nanopores in their hand-held sensing devices, which are capable of monitoring the outputs of hundreds of pores in parallel, offering the prospect of step changes in sensing technology in areas including biological warfare defense, food authentication, plant and animal breeding and medical diagnostics.

Technical Summary

Stochastic sensing with nanopores is a versatile single-molecule technology that can be used for the recognition and quantification of a wide range of analytes. Protein pores are advantageous in this respect, because they can be engineered with atomic precision and prepared in near homogeneous form. Until now, narrow protein pores have been used and therefore stochastic sensing has been confined to analytes of low mass or, in the case of nanopore DNA sequencing, to extended polymer chains. Here, we propose to make a new class of functional membrane-spanning nanopores, DNA-Protein hybrid nanopores, from DNA scaffolds to which peptide chains (either beta strands or alpha helices) are attached by bioorthogonal chemistry to form transmembrane barrel domains. This will be the first approach capable of producing pores of large internal diameter (5-30 nm) that are monodisperse and capable of precise site-specific modification: each pore will have an identical, predetermined number of subunits and no incomplete pores will be present. The DNA-protein hybrid nanopores will have several applications, for example to extend the scope of stochastic sensing to macromolecular analytes, e.g. for the recognition and quantification of large folded proteins, such as enzymes, receptors and antibodies. Our industrial partner is Oxford Nanopore Technologies, a company that exploits new developments in single-molecule sensing. Oxford Nanopore will evaluate and test our most promising DNA-Protein hybrid nanopores in their hand-held sensing devices, which are capable of monitoring the outputs of hundreds of pores in parallel.

Planned Impact

We envisage impact in five areas: (i) Academia; (ii) Training; (iii) the Research environment; (iv) the Economy; (v) Public engagement, and we summarize highlights here. From an academic viewpoint, the insight into a completely unexplored area of macromolecular engineering is of considerable interest to the wider community. From a practical viewpoint, the work will provide a new class of nanopores, which will allow large analytes, such as proteins, to be detected by stochastic sensing. This application will in turn require experimental exploration of fundamental issues, such as the behaviour of folded macromolecules in confinement. PDRAs taking part in the proposed work will be trained rigorously from a modern multidisciplinary perspective, at an internationally competitive level, in work requiring molecular engineering, cutting edge biophysics and high-level data analysis. They will be expected to plan and complete sub-projects, rather than merely participate in them. The researchers will also gain transferable skills through the demanding reporting and continuing education arrangements of the HB and SH groups, and courses provided by Oxford and UCL. The project will enhance the research environment at the group, local and collaborative level, as well as in a broader context. For example, we will welcome additional participants in the project area from the international research community. With this in mind, our results will be disseminated and discussed at meetings worldwide, and our materials and data will be freely available. The project is an Industrial Partnership Award in conjunction with Oxford Nanopore Technologies, a leader in single-molecule detection, which has committed £200,000 to the endeavour. The PDRAs will gain industrial experience through frequent visits to the company. Oxford Nanopore will explore applications of the best examples of our hybrid pores. They will be tested in their portable devices, which are capable of monitoring hundreds of pors in parallel. Therefore, there is a strong likelihood that our efforts will result in commercial products for personalised diagnosis, environmental protection, food testing or defense against bioterrorism. Both HB and SH enjoy and have significant experience in public engagement. They will continue to connect with the public through web sites, open days, visits to schools, and public lectures.


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Description The assembly of peptides into membrane-spanning nanopores might be promoted by scaffolds to pre-organize the structures. Such scaffolds could enable the construction of uniform pores of various sizes and pores with controlled permutations around a central axis. Here, we show that DNA nanostructures can serve as scaffolds to arrange peptides derived from the octameric polysaccharide transporter Wza to form uniform nanopores in planar lipid bilayers. Our ring-shaped DNA scaffold is assembled from short synthetic oligonucleotides that are connected to Wza peptides through flexible linkers. When scaffolded, the Wza peptides form conducting nanopores of which only octamers are stable and of uniform conductance. Removal of the DNA scaf- fold by cleavage of the linkers leads to a rapid loss of the nanopores from the lipid bilayer, which shows that the scaffold is essential for their stability. The DNA scaffold also adds functionality to the nanopores by enabling reversible and permanent binding of complementary tagged oligonucleotides near the nanopore entrance.
Exploitation Route To enable the formation of uniform nanopores with various numbers of subunits, our scaffolds might also be used to direct the assembly of more promiscuous peptides, either designed de novo or derived from antimicrobial peptides. The ability to form uniform large nanopores would open an immense range of possibilities, from the detection and fingerprinting of proteins in complex mixtures to single-molecule studies of enzymes and the mapping of epigenetic markers on long strands of double-stranded DNA (dsDNA). DNA scaffolds could be used further to form well-defined assemblies of multiple membrane proteins and pores, each held on a different arm or on separate, linked scaffolds. To prevent degradation of these scaffolds in living cells, synthetic analogues of DNA, such as xeno nucleic acid, could be used. Finally, the control over the relative position of each oligonucleotide in our scaffold will allow the assembly of well-known nanopores, such as a-haemolysin, with specified permutations of the subunits around a central axis as a first step towards creating advanced artificial enzymes.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology