Single molecule covalent chemistry and catalysis

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

Abstract

Our research group has devised a way to observe the making and breaking of the chemical bonds that hold individual molecules together. Normally, bond-making and breaking reactions are studied in test tubes containing many millions of molecules. However, several aspects of chemical reactions cannot be observed by this means: for example, the existence of intermediates lasting a few thousandths of a second is often obscured. Our method employs a nanoreactor so small that it contains only about 400 water molecules as well as the single molecule undergoing the chemistry of interest. By observing the change in a tiny electrical current passing through the nanoreactor, we have been able to see, for the first time, sequential steps in complex reaction sequences one molecule at a time. The reactions we have chosen to investigate in the proposed research are important in human medicine and have been connected with cancer, cardiovascular problems, inflammation, sexual dysfunction and both accidental and deliberate poisoning. As well as contributing to our fundamental understanding of medical problems, the research we will perform permits the sensing of trace amounts of material based on chemical reactivity, including molecules found in chemical weapons, pesticides, foodstuffs and pharmaceuticals. This detection technology may form the basis for a new generation of commercial sensing equipment.

Technical Summary

The investigation of the making and breaking of covalent bonds at the single molecule level has fundamental implications for mechanistic biological chemistry, enzymatic catalysis and the development of sensors for reactive molecules of physiological interest. In this programme, a new way to observe the chemistry of individual small molecules in aqueous solution is developed in which a protein pore is used as a nanoreactor . Electrical measurements in a bilayer apparatus are used to observe the chemistry of molecules at sites inside the pore. The ionic current carried by the pore in an applied potential is highly sensitive to the size, shape and charge of internal reactants. Hence, molecular rearrangements and bond formation and cleavage can be monitored. The single molecule approach has several advantages over ensemble measurements, notably the ability to observe short-lived intermediates, including those that arise after a slow step, and the ability to untangle complex reaction pathways. The use of electrical recording has advantages over alternative single molecule techniques. The experiments are done at dynamic equilibrium so that rate constants can be measured in the microsecond time domain without complications, such as a need for mixing. All the usual experimental variables (temperature, pH, ionic strength, etc.) are well controlled. There is no need for a spectroscopic probe, such as a fluorophore, that might perturb the reaction under observation. The reactions we have chosen to investigate are important in biology. For example, disulfide bond formation is important in protein folding and redox signaling. Nitrosothiols are involved in the nitric oxide signaling pathway and their chemistry remains controversial. Arsenic chemistry is important in pharmacology and toxicology. The work will also shed light on fundamental principles of catalysis and in the long-term may lead to new catalytic processes, for example, simultaneous substrate conversion and transmembrane translocation. Finally, we have been developing stochastic sensing , in which single molecule detection is used to determine the identity and concentration of physiological analytes. The work described here will provide information required for the detection of molecules found in chemical weapons, pesticides, foodstuffs and pharmaceuticals based on their chemical reactivity.

Publications

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Banerjee A (2010) Molecular bases of cyclodextrin adapter interactions with engineered protein nanopores. in Proceedings of the National Academy of Sciences of the United States of America

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Boersma AJ (2012) Continuous stochastic detection of amino acid enantiomers with a protein nanopore. in Angewandte Chemie (International ed. in English)

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Chen M (2008) Outer membrane protein G: Engineering a quiet pore for biosensing. in Proceedings of the National Academy of Sciences of the United States of America

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Chen M (2008) Orientation of the monomeric porin OmpG in planar lipid bilayers. in Chembiochem : a European journal of chemical biology

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Choi LS (2012) S-nitrosothiol chemistry at the single-molecule level. in Angewandte Chemie (International ed. in English)

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Das SK (2007) Membrane protein stoichiometry determined from the step-wise photobleaching of dye-labelled subunits. in Chembiochem : a European journal of chemical biology

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Hammerstein AF (2010) Single-molecule kinetics of two-step divalent cation chelation. in Angewandte Chemie (International ed. in English)

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Hammerstein AF (2011) Subunit dimers of alpha-hemolysin expand the engineering toolbox for protein nanopores. in The Journal of biological chemistry

 
Description ERC Advanced Investigator
Amount £2,250,000 (GBP)
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start 04/2012 
End 03/2017
 
Title Droplet interface bilayers 
Description This report refers to the year 2007. During this year (as reported under publications), we published our initial studies of droplet interface bilayers (DIB). For a recent review of this area see: Bayley, H., et al. Droplet interface bilayers, Mol. BioSystems 4, 1191-1208 (2008). DOI: 10.1039/b808893d. We found that aqueous droplets in oil containing lipid bind tightly to each other when they are brought together. A lipid bilayer is formed at the interface, with properties closely similar to the bilayers in lipid vesicles or the planar bilayers formed across small apertures. The DIB have numerous potential applications, which are presently under development. For example, when a bilayer is formed between two droplets, an electrode can be inserted into each one. If a membrane channel or pore is present in the DIB, the ionic current through the pore can be recorded when a potential is applied across the bilayer (PMID: 17571891). This process uses far smaller volumes than conventional bilayer recording, suggesting that it might be scaled up for use in the screening of medically relevant ion channels. In a second application, we found that several or many droplets can be formed into a network with a bilayer at the interface of each component. When channels or pores are present in the bilayers the array of droplets acts like an electrical circuit (PMID: 17764183). These results that suggest that networks might be made that mimic several aspects of biological tissues. 
Type Of Material Technology assay or reagent 
Year Produced 2007 
Provided To Others? Yes  
Impact We contimue to develop this area: Bayley, H., et al. Droplet interface bilayers, Mol. BioSystems 4, 1191-1208 (2008). DOI: 10.1039/b808893d. 
URL http://europepmc.org/abstract/MED/17764183
 
Description Engineered protein pores in bionanotechnology 
Organisation Oxford Nanopore Technologies
Country United Kingdom 
Sector Private 
PI Contribution Oxford Nanopore Technologies (ONT) support four postdoctoral research associates in my laboratory. While these PDRAs do not work directly on MRC projects, the work in my laboratory is highly interactive. Hence, ONT postdocs have contributed to MRC projects and vice-versa.
Collaborator Contribution Oxford Nanopore Technologies (ONT) support four postdoctoral research associates in my laboratory. While these PDRAs do not work directly on MRC projects, the work in my laboratory is highly interactive. Hence, ONT postdocs have contributed to MRC projects and vice-versa.
Impact Oxford Nanopore Technologies (ONT) support four postdoctoral research associates in my laboratory. While these PDRAs do not work directly on MRC projects, the work in my laboratory is highly interactive. Hence, ONT postdocs have contributed to MRC projects and vice-versa.
 
Title A transmembrane protein pore suitable for use in detecting an analyte in a sample 
Description Molecular adaptors A transmembrane protein pore suitable for use in detecting an analyte in a sample, comprising: a molecular adaptor that facilitates an interaction between the pore and the analyte, wherein the adaptor is covalently attached to the pore in an orientation that allows the analyte to be detected using the pore. The molecular adaptor is preferably a cyclodextrin, specifically heptakis-6-amino- b -cyclodextrin (am7- b -CD) or 6-monodeoxy-6-monoamino- b -cyclodextrin (am1 b CD); the covalent attachment is preferably via a bifunctional crosslinker; and the analyte is preferably an individual nucleotide. Methods of: producing a transmembrane protein pore; determining the presence or absence of an analyte; identifying an individual nucleotide; sequencing a target nucleic acid sequence in conjunction with a processive exonuclease are claimed, as is a kit for sequencing a nucleic acid. 
IP Reference EP2198046 
Protection Patent granted
Year Protection Granted 2010
Licensed Yes
Impact IP has been licensed by Oxford Nanopore Technologies
 
Title FORMATION OF BILAYERS OF AMPHIPATHIC MOLECULES 
Description A method of forming bilayers of amphipathic molecules uses droplets of aqueous solution in a hydrophobic medium such as oil. A layer of amphipathic molecules such as a lipid is formed around the surfaces of the droplets. This may be achieved by providing the lipid in the oil and leaving the droplets for a time sufficient to form the layer. The droplets are brought into contact with one another so that a bilayer of the amphipathic molecules is formed as an interface between the contacting droplets. The bilayers may be used for a wide range of studies. The technique has numerous advantages including providing a long lifetime for the bilayers, allowing study of small volumes and allowing the construction of chains and networks of droplets with bilayers in between to study complex systems. 
IP Reference WO2008012552 
Protection Patent granted
Year Protection Granted 2008
Licensed Commercial In Confidence
Impact IP has been licensed by Oxford Nanopore Technologies
 
Title METHODS OF ENHANCING TRANSLOCATION OF CHARGED ANALYTES THROUGH TRANSMEMBRANE PROTEIN PORES 
Description The invention relates to enhancing translocation of a charged analyte through a transmembrane protein pore. Translocation is enhanced by increasing the net opposing charge of the barrel or channel and/or entrance of the pore. The invention also relates to pores enhanced in accordance with the invention. 
IP Reference WO2010055307 
Protection Patent granted
Year Protection Granted 2010
Licensed Commercial In Confidence
Impact IP has been licensed by Oxford Nanopore Technologies
 
Title METHODS USING PORES 
Description The invention relates to a method of identifying an individual nucleotide, comprising (a) contacting the nucleotide with a transmembrane protein pore so that the nucleotide interacts with the pore and (b) measuring the current passing through the pore during the interaction and thereby determining the identity of the nucleotide. The invention also relates to a method of sequencing nucleic acid sequences and kits related thereto. Also published as WO2007057668 (A1) US2008311582 (A1) RU2008123842 (A) JP2009519705 (T) EP1951898 (A1) CN101356288 (A) CA2629009 (A1)AU2006314296 (A1) 
IP Reference KR20080083281 
Protection Patent granted
Year Protection Granted 2008
Licensed Commercial In Confidence
Impact IP has been licensed by Oxford Nanopore Technologies
 
Title Method for sequencing a heteropolymeric target nucleic acid 
Description The invention relates to a method for sequencing a heteropolymeric target nucleic acid sequence that involves stochastic sensing. The invention also relates to a method for improving a pore for sequencing a target nucleic acid sequence by modifying one or more sites in the pore. 
IP Reference WO2010109197 
Protection Patent granted
Year Protection Granted 2010
Licensed Commercial In Confidence
Impact IP has been licensed by Oxford Nanopore Technologies
 
Title Method of delivering a molecule to a lipid bilayer 
Description A method of delivering a molecule, such as a membrane protein, to a lipid bilayer (9) uses a probe (10) capable of holding the molecule on a carrier surface (15) thereof. The molecule is deposited on the carrier surface (15) and the probe (10) is moved to engage the carrier surface (15) against the lipid bilayer (9). The carrier surface (15) may be the surface of a drop of hydrogel (14) which adsorbs the molecule. The molecule may be a membrane protein which is thus inserted into the lipid bilayer (9). The method is fast and simple to perform thereby allowing high throughput experimentation. 
IP Reference US7939270 
Protection Patent granted
Year Protection Granted 2011
Licensed Yes
Impact IP has been licensed by Oxford Nanopore Technologies