Enzyme-Pore Constructs
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(19) TZZ Z_T (11) EP 2 682 460 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 08.01.2014 Bulletin 2014/02 C12N 9/12 (2006.01) C12N 9/22 (2006.01) C12N 9/52 (2006.01) C12Q 1/68 (2006.01) (21) Application number: 13187149.3 (22) Date of filing: 06.07.2009 (84) Designated Contracting States: • Cheley, Stephen AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Oxford HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL Oxfordshire OX4 4GA (GB) PT RO SE SI SK SM TR •McKeown,Brian Oxford (30) Priority: 07.07.2008 US 78695 P Oxfordshire OX4 4GA (GB) • White, James (62) Document number(s) of the earlier application(s) in Oxford accordance with Art. 76 EPC: Oxfordshire OX4 4GA (GB) 09784644.8 / 2 307 540 • Clarke, James Oxford (71) Applicant: Oxford Nanopore Technologies Oxfordshire OX4 4GA (GB) Limited Oxford Science Park (74) Representative: Chapman, Lee Phillip Oxford J A Kemp OX4 4GA (GB) 14 South Square Gray’s Inn (72) Inventors: London WC1R 5JJ (GB) • Jayasinghe, Lakmal Oxford Remarks: Oxfordshire OX4 4GA (GB) This application was filed on 02-10-2013 as a •Bayley,Hagan divisional application to the application mentioned Oxford under INID code 62. Oxfordshire OX4 4GA (GB) (54) Enzyme-pore constructs (57) The invention relates to constructs comprising a transmembrane protein pore subunit and a nucleic acid handling enzyme. The pore subunit is covalently at- tached to the enzyme such that both the subunit and enzyme retain their activity. The constructs can be used to generate transmembrane protein pores having a nu- cleic acid handling enzyme attached thereto. Such pores are particularly useful for sequencing nucleic acids. The enzyme handles the nucleic acid in such a way that the pore can detect its component nucleotides by stochastic sensing. EP 2 682 460 A1 Printed by Jouve, 75001 PARIS (FR) EP 2 682 460 A1 Description Field of the invention 5 [0001] The invention relates to constructs comprising a transmembrane protein pore subunit and a nucleic acid handling enzyme. The pore subunit is covalently attached to the enzyme such that both the subunit and enzyme retain their activity. The constructs can be used to generate transmembrane protein pores having a nucleic acid handling enzyme attached thereto. Such pores are particularly useful for sequencing nucleic acids. The enzyme handles the nucleic acid in such a way that the pore can detect each of its component nucleotides by stochastic sensing. 10 Background of the invention [0002] Stochastic detection is an approach to sensing that relies on the observation of individual binding events between analyte molecules and a receptor. Stochastic sensors can be created by placing a single pore of nanometer 15 dimensions in an insulating membrane and measuring voltage-driven ionic transport through the pore in the presence of analyte molecules. The frequency of occurrence of fluctuations in the current reveals the concentration of an analyte that binds within the pore. The identity of an analyte is revealed through its distinctive current signature, notably the duration and extent of current block (Braha, O., Walker, B., Cheley, S., Kasianowicz, J. J., Song, L., Gouaux, J. E., and Bayley, H. (1997) Chem.Biol. 4, 497-505; and Bayley, H., and Cremer, P. S. (2001) Nature 413, 226-230). 20 [0003] Engineered versions of the bacterial pore forming toxinα -hemolysin (α-HL) have been used for stochastic sensing of many classes of molecules (Bayley, H., and Cremer, P. S. (2001) Nature 413, 226-230; Shin, S., H., Luchian, T., Cheley, S., Braha, O., and Bayley, H. (2002) Angew.Chem.Int.Ed. 41, 3707-3709; and Guan, X., Gu, L.-Q., Cheley, S., Braha, O., and Bayley, H. (2005) ChemBioChem 6, 1875-1881). In the course of these studies, it was found that attempts to engineer α-HL to bind small organic analytes directly can prove taxing, with rare examples of success (Guan, 25 X., Gu, L.-Q., Cheley, S., Braha, O., and Bayley, H. (2005) ChemBioChem 6, 1875-1881). Fortunately, a different strategy was discovered, which utilized non- covalently attached molecular adaptors, notably cyclodextrins (Gu, L.-Q., Braha, O., Conlan, S., Cheley, S., and Bayley, H. (1999) Nature 398, 686-690), but also cyclic peptides (Sanchez-Quesada, J., Ghadiri, M. R., Bayley, H., and Braha, O. (2000) J.Am.Chem.Soc. 122, 11758-11766) and cucurbiturils (Braha, O., Webb, J., Gu, L.-Q., Kim, K., and Bayley, H. (2005) ChemPhysChem 6, 889-892). Cyclodextrins become transiently 30 lodged in the α-HL pore and produce a substantial but incomplete channel block. Organic analytes, which bind within the hydrophobic interiors of cyclodextrins, augment this block allowing analyte detection (Gu, L.-Q., Braha, O., Conlan, S., Cheley, S., and Bayley, H. (1999) Nature 398, 686-690). [0004] There is currently a need for rapid and cheap DNA or RNA sequencing technologies across a wide range of applications. Existing technologies are slow and expensive mainly because they rely on amplification techniques to 35 produce large volumes of nucleic acid and require a high quantity of specialist fluorescent chemicals for signal detection. Stochastic sensing has the potential to provide rapid and cheap DNA sequencing by reducing the quantity of nucleotide and reagents required. Summary of the invention 40 [0005] The inventorshave surprisingly demonstratedthat covalent attachment of atransmembrane protein poresubunit to a nucleic acid handling enzyme results in a construct that is capable of both forming a pore and handling nucleic acids. The inventors have also surprisingly demonstrated that the construct can be used to generate a transmembrane protein pore that is capable of both handling a nucleic acid and sequencing the nucleic acid via stochastic sensing. The 45 fixed nature and close proximity of the enzyme to the pore means that a proportion of the nucleotides in a target nucleic acid will interact with the pore and affect the current flowing through the pore in a distinctive manner. As a result, transmembrane protein pores comprising such constructs are useful tools for stochastic sensing and especially for sequencing nucleic acids. [0006] Accordingly, the invention provides a construct comprising a transmembrane protein pore subunit and a nucleic 50 acid handling enzyme, wherein the subunit is covalently attached to the enzyme, wherein the subunit retains its ability to form a pore and wherein the enzyme retains its ability to handle nucleic acids. The invention also provides: - a polynucleotide sequence which encodes a construct of the invention; - a modified pore for use in sequencing nucleic acids, comprising at least one construct of the invention; 55 - a kit for producing a modified pore for use in sequencing nucleic acids, comprising: (a) at least one construct of the invention; and (b) any remaing subunits needed to form a pore; 2 EP 2 682 460 A1 - a kit for producing a modified pore for use in sequencing nucleic acids, comprising: (b) at least one polynucleotide of the invention; and (c) poynucleotide sequences encoding any remaining subunits needed to form a pore; 5 - a method of producing a construct of the invention, comprising: (a) covalently attaching a nucleic acid handling enzyme to a transmembrane protein pore subunit; and (b) determining whether or not the resulting construct is capable of forming a pore and handling nucleic acids; 10 - a method of producing a modified pore of the invention, comprising: (a) covalently attaching a nucleic acid handling enzyme to a transmembrane protein pore; and (b) determining whether or not the resulting pore is capable of handling nucleic acids and detecting nucleotides; 15 - method of producing a modified pore of the invention, comprising: (a) allowing at least one construct of the invention to form a pore with other suitable subunits; and (b) determining whether or not the resulting pore is capable of handling nucleic acids and detecting nucleotides. 20 - a method of purifying a transmembrane pore comprising at least one construct of the invention, comprising: (a) providing the at least one construct and the other subunits required to form the pore; (b) oligomerising the at least one construct and other subunits on synthetic lipid vesicles; and 25 (c) contacting the vesicles with a non-ionic surfactant; and (d) recovering the oligomerised pore; - a method of sequencing a target nucleic acid sequence, comprising: 30 (a) contacting the target sequence with a pore of the invention, which comprises an exonuclease, such that the exonuclease digests an individual nucleotide from one end of the target sequence; (b) contacting the nucleotide with the pore so that the nucleotide interacts with the adaptor; (c) measuring the current passing through the pore during the interaction and thereby determining the identity of the nucleotide; and 35 (d) repeating steps (a) to (c) at the same end of the target sequence and thereby determining the sequence of the target sequence; and - a method of sequencing a target nucleic acid sequence, comprising: 40 (a) contacting the target sequence with a pore of the invention so that the enzyme pushes or pulls the target sequence through the pore and a proportion of the nucleotides in the target sequence interacts with the pore; and (b)measuring the current passing through thepore during eachinteraction and thereby determining the sequence of the target sequence. 45 Description of the Figure [0007] Figure 1 shows how exonuclease enzymes catalyse the hydrolysis of phosphodietser bonds. Within the active site 50 of the exonulease, a water molecule is enabled to react with the phosphate of the 3’ end of the polynucleotide (DNA). Cleavage of the bond between the phosphate and the sugar towards the 5’ end releases a monophosphate (deoxy) nucleoside. Figure 2 shows the crystal structures of exonucleases used in the Example, N and C- terminus and active sites are shown for each.