Minimal DNA Nanopores for Electrical Sensing of Proteins

Lead Research Organisation: University College London
Department Name: Chemistry

Abstract

Proteins are of paramount importance in our lives. They carry out the main functions in our bodies and control our movement, energy conversion, immune defence, and thinking. In medicine, functionally aberrant proteins cause disease. The proteins can, however, be targeted by drugs to cure cells. Enzymes are also of biotechnological importance in the cost-efficient synthesis of drug molecules or for the energy-saving cleaning of fabrics.

Detecting and analysing proteins is the first step towards predicting diseases, developing cures, and engineering proteins for industry. The analysis of proteins improves our understanding their structure, dynamics, and function. Ideally, sensing of proteins should be simple and fast and be conducted using inexpensive and portable equipment. This increase research efficiency and opens up point-of-care sensing in diagnosis and homeland security.

This project will provide a new way to sense proteins in a portable yet scientifically accurate fashion thereby overcoming problems of existing approaches. Classical approaches have issues such as the requirement to label the proteins with a fluorescent tag which can interfere with the structure and function of the proteins. Optical detection can also increase the weight and cost of the analytical device. Another limitation of conventional sensing approaches is that they average over millions of molecules and have difficulties to detect biologically important sub-groups.

We will develop a new approach to sense proteins in a label and optics-free electrical fashion using portable equipment capable of uncovering proteins down to the level of individual molecules. The proposed strategy is currently being used for DNA strand sequencing. Our industry partner Oxford Nanopore Technologies has developed a hand-held device for genome sequencing. The analytes are detected when individual strands pass through nanoscale pores in a thin membrane. The temporary blockade of the pores alters the electronic read-out signal similar to the reduction of water flow when a stone is inside a tube.

We will be able to sense proteins which are wide enough to accommodate proteins. The new pores will be composed of DNA stands, thereby exploiting the exquisite ability of DNA to function as a nanoscale construction material. Chemical modification will be key to achieve the functional performance of the pores. To maximise the benefit for academia, industry and society, we will strongly collaborate with our commercial partner to test the new pores in the portable electrical sensing devices.

Planned Impact

The project will generate biosensor components composed of DNA nanopores which will enable the electrical and label-free sensing of proteins of biomedical or biosafety relevance in research and in point-of-care settings.

The first impact of DNA nanopores will be in basic science and single-molecule research. By enabling label-free real-time sensing in solution, the approach will avoid the drawbacks of other competing strategies which require fluorescence labeling or immobilization at an inorganic surface (AFM) which can interfere with the protein's natural confirmation. The nanopore approach is compatible with a wide range of analytes including enzymes, signaling proteins, and immune-relevant proteins. The DNA nanopores are termed minimal as they are reduced to the essential functional parts.

The second impact will be in diagnostic sensing using devices from industry partner Oxford Nanopore Technologies. DNA nanopores can expand the scope of analytes detected with electrical sensing to biomedically relevant proteins including immunoproteins or enzymes that interact with drugs. The collaboration with ONT will ensure that the nanopores will be tested in state-of-the-art analysis platforms. Feedback will be provided on the performance of the DNA nanopores in commercial biosensing.

The proposal is, thrid, of national importance as it will strengthen the industrial base by helping develop new sensor components for companies such as ONT. By expanding the scope of nanopore sensing, the proposal will also add to UK's academic and economic strength in this area.

Publications

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Arnott PM (2018) Dynamic Interactions between Lipid-Tethered DNA and Phospholipid Membranes. in Langmuir : the ACS journal of surfaces and colloids

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Burns JR (2019) Structural and Functional Stability of DNA Nanopores in Biological Media. in Nanomaterials (Basel, Switzerland)

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Diederichs T (2019) Synthetic protein-conductive membrane nanopores built with DNA. in Nature communications

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Dorey A (2023) Unfolding the path to nanopore protein sequencing. in Nature nanotechnology

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Howorka S (2017) Building membrane nanopores. in Nature nanotechnology

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Howorka S (2016) NANOTECHNOLOGY. Changing of the guard. in Science (New York, N.Y.)

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Lanphere C (2022) Triggered Assembly of a DNA-Based Membrane Channel. in Journal of the American Chemical Society

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Lanphere C (2021) A Biomimetic DNA-Based Membrane Gate for Protein-Controlled Transport of Cytotoxic Drugs. in Angewandte Chemie (International ed. in English)

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Maingi V (2017) Stability and dynamics of membrane-spanning DNA nanopores. in Nature communications

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Messager L (2016) Biomimetic Hybrid Nanocontainers with Selective Permeability. in Angewandte Chemie (International ed. in English)

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Messager L (2016) Biomimetic Hybrid Nanocontainers with Selective Permeability in Angewandte Chemie

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Nguyen HP (2019) A Photo-responsive Small-Molecule Approach for the Opto-epigenetic Modulation of DNA Methylation. in Angewandte Chemie (International ed. in English)

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Offenbartl-Stiegert D (2019) Solvent-dependent photophysics of a red-shifted, biocompatible coumarin photocage in Organic & Biomolecular Chemistry

 
Description Proteins are of paramount importance in our lives. They carry out the main functions in our bodies and control our movement, energy conversion, immune defence, and thinking. In medicine, functionally aberrant proteins cause disease. The proteins can, however, be targeted by drugs to cure cells. Enzymes are also of biotechnological importance in the cost-efficient synthesis of drug molecules or for the energy-saving cleaning of fabrics.

Detecting and analysing proteins is the first step towards predicting diseases, developing cures, and engineering proteins for industry. The analysis of proteins improves our understanding their structure, dynamics, and function. Ideally, sensing of proteins should be simple and fast and be conducted using inexpensive and portable equipment. This increase research efficiency and opens up point-of-care sensing in diagnosis and homeland security.

This project has helped create a new way to sense proteins in a portable yet scientifically accurate fashion thereby overcoming problems of existing approaches. Classical approaches have issues such as the requirement to label the proteins with a fluorescent tag which can interfere with the structure and function of the proteins. Optical detection can also increase the weight and cost of the analytical device. Another limitation of conventional sensing approaches is that they average over millions of molecules and have difficulties to detect biologically important sub-groups.

We have developed a new approach to sense proteins in a label and optics-free electrical fashion using portable equipment capable of uncovering proteins down to the level of individual molecules. The proposed strategy is currently being used for DNA strand sequencing. Our industry partner Oxford Nanopore Technologies sells a hand-held device for genome sequencing. The analytes are detected when individual strands pass through nanoscale pores in a thin membrane. The temporary blockade of the pores alters the electronic read-out signal similar to the reduction of water flow when a stone is inside a tube.

In this project, we have developed a route to sense proteins which are wide enough to accommodate proteins. The new pores are composed of DNA stands, thereby exploiting the exquisite ability of DNA to function as a nanoscale construction material. Chemical modification help achieve the functional performance of the pores.
Exploitation Route The industrial partner biotechnology company Oxford Nanopore Technologies might use the sensor pores created in this and another project for developing portable sensing devices for point-of-care diagnostics, environmental screening or homeland security.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description The work on the DNA nanopore, which is the topic of the research project, has led to IP which has been licensed to the company which is leading portable sequencing and sensing with nanopores, Oxford Nanopore Technologies. The PI of the project and the company are currently working on improving the DNA nanopore technology in order to bring it to market as sensing product.
Sector Electronics,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Research Project Grants
Amount £149,186 (GBP)
Funding ID RPG-2017-015 
Organisation The Leverhulme Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2018 
End 02/2020
 
Title MEMBRANE BOUND NUCLEIC ACID NANOPORES 
Description A membrane-spanning nanopore is provided, the nanopore comprising: a. one or more polynucleotide strands that provide a scaffold component; and b. a plurality of polynucleotide strands that provide a plurality of staple components; wherein each of the plurality of staple components hybridise to the scaffold component; wherein the orientation of a major portion of at least one polynucleotide strand comprised within the scaffold component is substantially parallel to a planar surface of a membrane as well as embedded within and substantially coplanar with the membrane; wherein the nanopore defines a channel that is suitable for perforating the membrane; the channel having a longitudinal axis extending along the centre thereof and a minimum internal dimension perpendicular to the longitudinal axis of at least about 3 nm. A membrane comprising the nanopore, biological sensors comprising the membrane and a method for molecular sensing using the membrane are also disclosed 
IP Reference WO2020025974 
Protection Patent application published
Year Protection Granted 2020
Licensed Yes
Impact The patent has been licensed by the industrial partner of the grant.
 
Title MEMBRANE-SPANNING NANOPORES 
Description A membrane-spanning nanopore is provided that comprises:i. at least one scaffold polynucleotide strand; ii. a plurality of staple polynucleotide strands; and iii. at least one hydrophobically-modified polynucleotide strand, wherein the at least one hydrophobically-modified polynucleotide strand comprises a polynucleotide strand and a hydrophobic moiety;wherein each of the plurality of staple polynucleotide strands hybridises to the at least one scaffold polynucleotidestrand to form the three-dimensional structure of the membrane-spanning nanopore, and wherein the at least one hydrophobically-modified polynucleotide strand hybridises to a portion of the at least one scaffold polynucleotide strand, the membrane-spanning nanopore defining a central channel with a minimum internal width of at least about 5 nm. Membranes comprising the membrane-spanning nanopore and applications of those membranes are also provided. 
IP Reference WO2018011603 
Protection Patent application published
Year Protection Granted 2018
Licensed Yes
Impact The work in the project has led to a patent application which is being licensed by biotech company Oxford Nanopore Technologies. The patent is entitled MEMBRANE-SPANNING NANOPORES, PCT/GB2017/052089, priority date 14 July 2017. A second patent on DNA nano pores has been filed and licensed by Oxford Nanopore Technologies. The second application is "Membrane Bound Nucleic Acid Nanopores" PATENT APPLICATION NUMBER 1812615.1, priority data 2 Aug 2018.