WO2009007743A1 - Procédé de détection d'acides nucléiques - Google Patents

Procédé de détection d'acides nucléiques Download PDF

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Publication number
WO2009007743A1
WO2009007743A1 PCT/GB2008/050536 GB2008050536W WO2009007743A1 WO 2009007743 A1 WO2009007743 A1 WO 2009007743A1 GB 2008050536 W GB2008050536 W GB 2008050536W WO 2009007743 A1 WO2009007743 A1 WO 2009007743A1
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nucleic acid
membrane
acid molecule
primer
bases
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PCT/GB2008/050536
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English (en)
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Stefan Howorka
Nick Mitchell
Vinciane Borsenberger
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Ucl Business Plc
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Publication of WO2009007743A1 publication Critical patent/WO2009007743A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores

Definitions

  • the invention relates to the use of single-channel current (nanopore) recordings in combination with chemically modified DNA to retrieve sequence-specific information.
  • the approach can be applied for detecting single nucleotide polymorphisms (SNPs), for expression profiling, and for the sizing of highly repetitive sequences of forensic and biomedical importance.
  • SNPs single nucleotide polymorphisms
  • Membrane protein channels and artificial nanopores provide the opportunity to detect analytes via electrical recordings.
  • an ionic current passes through the pores which fluctuates if the pores are partially or completely blocked by an analyte.
  • Such fluctuations in the current can be analysed to identify both the concentration and identity of an analyte, the latter from its distinctive current signature.
  • Stochastic sensing which uses currents from single pores, is an especially attractive prospect because it is highly sensitive and provides a rapid and reversible response which allows real-time monitoring of analytes.
  • Stochastic sensing has been used to detect ionic molecules, organic molecules and macro molecules such as single-stranded RNA and DNA.
  • a transmembrane potential drives individual DNA and RNA strands through a nanopore and an ionic current is at the same time driven through the pore by the applied potential.
  • an ionic current is at the same time driven through the pore by the applied potential.
  • DNA homopolymers of different composition give rise to different characteristic current blockades and modulations [J. J. Kasianowicz, E. Brandin, D. Branton, D. W.
  • US 2005/0053961 relates to the characterisation of polymers, such as DNA, by their interaction with a pore to bring about changes in conductance across the pore which are indicative of the characteristics of the polymer. It is suggested that the nucleotide bases of DNA will influence pore conductance during passage of a DNA molecule through a pore and the sensitivity of the system may be increased by using modified bases, such as methylated bases and biotinylated triphosphates.
  • SNPs single nucleotide polymorphisms
  • the invention in a first aspect relates to a method of detecting the presence or absence of one or more bases or the number of repeats of one or more bases in an analyte nucleic acid molecule, comprising providing a membrane having first and second sides and containing a nanoscale pore having a lumen spanning the membrane; providing a fluid comprising the analyte nucleic acid molecule and an ionic salt on the first side of the membrane and a solution comprising an ionic salt on the second side of the membrane; and applying a potential difference across the membrane and measuring the resulting current over time, wherein the analyte nucleic acid molecule is chemically modified to modulate the duration and/or amplitude of the measured current and thereby allow identification of the analyte nucleic acid molecule and detection of the presence or absence of the one or more bases or the number of repeats of the one or more bases.
  • the analyte nucleic acid molecule comprises an extended primer oligonucleotide consisting of an original primer portion and an extended oligonucleotide portion, which is chemically modified in the original primer portion and optionally in the extended oligonucleotide portion, the presence or extent of chemical modification in the extended oligonucleotide portion being related to the presence or absence of the one or more bases or the number of repeats of the one or more bases.
  • the invention in another aspect relates to a method of detecting the presence or absence of one or more bases or the number of repeats of one or more bases in an analyte nucleic acid molecule, comprising: providing a sample of nucleic acid molecules and a chemically modified primer complementary to a portion of the analyte nucleic acid molecule; allowing the primer to bind to analyte nucleic acid molecules in the sample and to extend wherein one or more chemically modified nucleotides are incorporated into the extended primer oligonucleotide when one or more complementary bases are present in the bound analyte nucleic acid molecule; providing a membrane having first and second sides and containing a nanoscale pore having a lumen spanning the membrane; providing a fluid comprising the chemically modified extended primer oligonucleotide and an ionic salt on the first side of the membrane and a solution comprising an ionic salt on the second side of the membrane; and applying a potential difference across the membrane and measuring the
  • the primer extension approach can be used for multiplexing to analyse multiple different SNPs in a biological sample.
  • the use of multiple DNA primers which are modified with different sequences of chemical tags which give rise to a characteristic pattern of current modulations is the key to achieve multiplexing. In this way, the sequence of the primer can be inferred from the characteristic pattern of current modulations in the traces. Detecting the signatures in the sample with the extended primer therefore indicates that a specific SNP must have been present in the original sample.
  • each type of chemical modification is associated with a specific base.
  • a single base may have one or more chemical modifications, but each chemical modification may be associated with only a single base and not two or more consecutive bases.
  • the chemical modification is smaller than 1.5 nm diameter, more preferably smaller than 1 nm diameter, more preferably smaller than 0.5 nm diameter and most preferably about 0.3 nm diameter.
  • the modification may act to increase the diameter of the nucleic acid molecule, thereby slowing its passage through the nanopore.
  • the chemical modification is a peptide tag, preferably consisting of from 2 to 6 amino acids.
  • the peptide tag may be selected from hexahistidine, hexaarginine, hexaaspartic acid, histidine(4), histidine(2) and tyrosine(3).
  • the primer may be modified with one or more chemical tags which are different to the one or more chemical tags used to detect the presence or absence of the one or more bases or the number of repeats of the one or more bases.
  • the chemical tag may also allow the extended primer oligonucleotide to be isolated from other nucleic acid molecules.
  • each type of base which is detected is labelled with a different chemical tag which gives rise to a specific current signature. Since it is possible to infer which type of base is present in the extend primer strand by looking at the current signature, this enables part or all of a nucleic acid molecule to be sequenced.
  • the invention provides a method of mRNA profiling comprising: providing a sample comprising a first mRNA species and a second mRNA species, a chemically modified first primer complementary to a portion of said first mRNA species and a chemically modified second primer complementary to a portion of said second mRNA species; allowing the primers to bind to the mRNA species in the sample and to extend wherein one or more chemically modified nucleotides is incorporated into the extended primer oligonucleotides when one or more complementary bases are present in the bound mRNA species; providing a membrane having first and second sides and containing a nanoscale pore having a lumen spanning the membrane; providing a fluid comprising the chemically modified extended primer oligonucleotides and an ionic salt on the first side of the membrane and a solution comprising an ionic salt on the second side of the membrane; and applying a potential difference across the membrane and measuring the resulting current over time, wherein the chemical modification of the extended primer oligonu
  • the chemical modification of the first primer is different to the chemical modification of the second primer and/or different chemically modified nucleotides are incorporated into the first and second mRNA species.
  • the invention provides a method of sequencing a nucleic acid molecule comprising: providing a sample comprising the nucleic acid molecule and a chemically modified primer complementary to a portion of said nucleic acid molecule; allowing the primer to bind to the nucleic acid molecule in the sample and to extend wherein one or more chemically modified nucleotides is incorporated into the extended primer oligonucleotide when one or more complementary bases are present in the bound nucleic acid molecule, wherein each type of chemically modified nucleotide is labelled with a different chemical tag which gives rise to a specific current signature; providing a membrane having first and second sides and containing a nanoscale pore having a lumen spanning the membrane; providing a fluid comprising the chemically modified extended primer oligonucleotides and an ionic salt on
  • Figure 1 shows the cross-sectional view of the heptameric ⁇ HL pore.
  • the model was generated using crystallographic data (Song, L.; Hobaugh, M. R.; Shustak, C; Cheley, S.; Bayley, H.; Gouaux, J. E. Science. 1996, 274, 1859-1866.) and PyMoI.
  • the internal diameters of the channel are: 2.9 nm, cis entrance; 4.1 nm, internal cavity; 1.3 nm, inner constriction; 2 nm, trans entrance of the ⁇ -barrel.
  • Figure 2 shows an individual nanopore embedded in a membrane which separates two reservoirs filled with electrolyte solution.
  • Figure 3 shows schematically a single channel current traces of (A) an unmodified synthetic oligonucleotide, (B) a synthetic oligonucleotide modified with one chemical tag covalently attached to a single base, (C) a synthetic oligonucleotide modified with a different chemical tag covalently attached to a single base, and (D) a synthetic oligonucleotide modified with two different chemical tags, each covalently attached to a single base.
  • Figure 4 shows the use of chemically modified DNA to detect an SNP using primer extension and representative single channel current traces.
  • 4A In the case of the presence of an SNP, a chemically modified nucleotide is incorporated resulting in an additional blockade signature.
  • 4B In the case of the absence of an SNP, no chemical tag is incorporated resulting in no additional blockade signature.
  • Figure 5 shows the use of chemically modified DNA to size the length of highly repetitive DNA strands.
  • 5 A In the case of the presence of two repeats, two chemically modified nucleotides are incorporated resulting in an additional blockade signature of length 2xt 2 .
  • 5B In the case of the presence of seven repeats, seven chemically modified nucleotides are incorporated resulting in an additional blockade signature of length 7xt 2 .
  • Figure 6 shows how the incorporation of chemically modified nucleotides can proceed in a two-step procedure.
  • a nucleotide with a small linker is incorporated efficiently into DNA.
  • the remaining chemical tag is attached to the linker.
  • Figure 7 shows single-channel current recordings for (A) a hexa-arginine modified oligonucleotide of 27 bases in length, (B) a hexa-histidine modified oligonucleotide of 27 bases in length, (C) an unmodified oligonucleotide of 27 bases in length, and (D) an oligonucleotide of 27 bases in length modified with two peptide tags, hexa-histine and tri-tyrosine.
  • Figure 8 shows (A) The ⁇ - hemolysin ( ⁇ HL) pore embedded in a lipid bilayer. (B) The chemical linkage between DNA and peptide within construct H6C1-O1. (C) Schematic representation of the ⁇ HL pore and a representative single channel current trace. (D) Events caused by oligonucleotide Ol without a peptide tag. (E) Trace for the translocation of H6C1-O1. (F) Events for H6C 1-01 -terminal. The traces were obtained from recordings at 2 M KCl, 20 mM Tris, pH 8.0, and filtered and sampled at 10 kHz and 50 kHz, respectively.
  • Figure 9 shows representative nanopore translocation events for (A) R7C1-O1, (B) Y3C1-O1 exhibiting a current step, (C) Y3C1-O1 exhibiting a current slope, and (D) Y3/Y3-O3.
  • the insets display the magnified view of the high-amplitude region of the corresponding events.
  • E Amino acid side chains or histidine, arginine and tyrosine.
  • F Scheme to account for the current signature of Y3/Y3-O3 events in (D).
  • an individual nanopore is embedded in a membrane which separates two reservoirs filled with electrolyte solution (see Fig. 2).
  • the pore can be a protein pore in a lipid bilayer membrane or a solid-state nanopore composed of Si 3 N 4 .
  • the application of a transmembrane potential leads to the flow of a small ionic current which can be measured using a current amplifier.
  • Nanopore recordings can be used to detect nucleic acid molecules, e.g. DNA or RNA, at the single molecule level.
  • nucleic acid molecules e.g. DNA or RNA
  • nucleic acid strands are electrophoretically driven through the pore as shown in Fig. 3A.
  • the temporary blockade of the pore leads to a reversible reduction in the current flowing through the pore.
  • nanopore recordings are capable of detecting individual nucleic acid strands.
  • the new approach uses chemically modified nucleic acid molecules in order to slow down the passage of the nucleic acid strand through the pore and to specifically detect individual bases.
  • the use of chemically modified DNA strands allows tuning of the cross-sectional diameter of ssDNA to existing pore dimensions rather than matching the pore dimensions to the size of the DNA strand.
  • one chemical tag was covalently attached to a single base in a synthetic oligonucleotide.
  • the chemical tag increases the cross-sectional area of the DNA to over 1 nm which is similar to the 1.2 nm wide nanopore. Recordings show that the chemical tag slows down the passage of the DNA strand by a factor of up to 15 leading to an average blockade duration tl (Fig.
  • the pore is an organic pore.
  • the term organic takes its usual meaning in the art, therefore the organic pore substantially comprises carbon and hydrogen, and also other elements, especially nitrogen, oxygen, sulfur, phosphorus and halogens. These pores exhibit very high mechanic stability which makes them ideally suited for rugged electrical sensor devices.
  • the pore is a protein pore.
  • a protein pore is a pore which is predominantly protein; however, other types of molecules may also be present.
  • Examples of protein pores suitable for use in the invention include alpha hemolysin, pneumolysin, outer membrane proteins such as porins, and other bacterial pore-forming toxins (Gilbert, R. J. (2002) Cell MoI Life Sci 59, 832-44) (Parker, M. W., and Feil, S. C. (2005) Prog Biophys MoI Biol 88, 91-142) such as streptolysin O (Bhakdi, S., Tranum- Jensen, J., and Sziegoleit, A.
  • the protein pore is a ⁇ -hemolysin ( ⁇ HL) polypeptide.
  • ⁇ HL is a bacterial toxin which self-assembles to form a heptameric protein pore.
  • the X-ray structure of the ⁇ HL pore resembles a mushroom with a wide cap and a narrow stem, which spans the lipid bilayer (Fig. 1) (Song, L.; Hobaugh, M. R.; Shustak, C; Cheley, S.; Bayley, H.; Gouaux, J. E. Science. 1996, 274, 1859-1866).
  • the external dimensions of the heptameric ⁇ HL pore are 10 x 10 nm, while the central channel is 2.9 nm in diameter at the cis entrance and widens to 4.1 nm in the internal cavity (Fig. 1). In the transmembrane region, the channel narrows to 1.3 nm at the inner constriction and broadens to 2 nm at the trans entrance of the ⁇ -barrel.
  • the defined structure of ⁇ HL has facilitated extensive engineering studies and has led to the development of tools for the targeted permeabilization of cells (Eroglu, A.; Russo, M. J.; Bieganski, R.; Fowler, A.; Cheley, S.; Bayley, H.; Toner, M. Nat Biotechnol. 2000, 18, 163-167) as well as new biosensor elements which permit the stochastic sensing of molecules (Bayley, H.; Cremer, P. S. Nature. 2001, 413, 226-230).
  • the invention is not limited to pores of this type.
  • the pore is an inorganic pore.
  • the inorganic pore is composed of silica, silicon nitride, alumina, titanium, gold, platinum, zirconia or a combination thereof.
  • Particularly preferred is a solid-state nanopore composed Of Si 3 N 4 .
  • the pore is not limited in relation to the material that it comprises, the invention is limited in relation to the size of the pore.
  • the pore must be a nanoscale pore.
  • nanoscale is meant that the pore is one wherein the lumen has a diameter of less than 1 ⁇ m.
  • the lumen has a diameter of less than 100 nm. More preferably the lumen has a diameter of 10 nm or less.
  • the pore has a diameter of at least 1 nm. References to the diameter of the pore are to be interpreted as the diameter of the pore at its minimum value. In this regard see fig. 1 which illustrates that the diameter of the lumen may vary at different positions along its length.
  • Engineered nanopores may also be used in accordance with the invention.
  • Engineered pores include pores which have been modified to affect the passage of molecules therethrough. The modification may be at the inner constriction or at either entrance.
  • the oligonucleotide has been modified with a positively charged chemical tag then it may be beneficial to use a protein nanopore which has been engineered to have negative residues in the inner constriction of the pore.
  • oligonucleotide passes through the pore there would be electrostatic interactions between the positively charged chemical tag and the negative residues in the lumen, which further slow the passage of the oligonucleotide through the pore and therefore specifically modulate the current.
  • electrostatic interactions other types of interactions are possible such as hydrogen bonding, van der Waals, steric bulk pi-pi interactions and hydrophobic interactions, metal chelate complex formation (e.g. NTA Ni HIS), secondary structures which have to be unfolded for their passage (e.g. hairpin structure).
  • the pores described above provide a route, channel or path across a membrane, from one side to the other side.
  • the membrane, or barrier itself is not limited in type, and would need to be chosen by consideration of species it is desired to pass through the pore or prevent from passing through the pore, and therefore between the membrane's first and second sides. For example, the membrane must be stable in the presence of this species.
  • membranes used in the method of the invention are not limited to biological materials: biological membranes are only one example of membranes according to the invention.
  • the membrane is organic.
  • the organic membrane is an organic polymer, most preferably the organic polymer is a polycarbonate or polyterephtalate polymer.
  • the organic membrane is a lipid bilayer.
  • the membrane comprising a pore is formed by allowing mutant polypeptides K46C, K8C or S106C to assemble on rabbit erythrocyte membranes to form heptameric pores.
  • the membrane is inorganic, wherein preferably the inorganic membrane is a gold-plated porous membrane prepared by the template synthesis method by depositing gold along the pore walls of a polycarbonate template membrane (Martin, C. R., Nishizawa, M., Jirage, K., and Kang, M. (2001) J Phys Chem B 105, 1925).
  • the pore is an organic pore, preferably a polymeric organic pore or a protein pore.
  • the membrane is an organic polymer the pore is an organic pore.
  • the membrane is an inorganic membrane, the pore is an inorganic pore.
  • the membrane is a lipid bilayer and the pore is a protein pore or a solid-state nanopore composed Of Si 3 N 4 .
  • the membrane will usually comprise a single pore, the lumen of which provides a single channel between the first side of the membrane and the second side of the membrane.
  • the use of single-molecule detection leads to a higher sensitivity. This could help reduce the number of PCR cycles and errors which are encountered in the course of the preparation of samples for microarray or bead- based detection schemes.
  • the membrane may also comprise more than one but less than ten pores provided that the current modulations caused by the translocation of a DNA strand through a first pore are not compromised by the current modulations caused by the translocation of another DNA strand through a second pore.
  • electrolytes may be used in accordance with the invention.
  • suitable electrolytes are KCl or NaCl in the concentration range from 0.5 M to 4 M, or organic electrolytes such as ammonium acetate.
  • the transmembrane potentials can vary from 30 to 200 mV in the case of lipid bilayer membranes but the upper boundary can be up to several V in the case of inorganic pores which are more robust.
  • either single stranded or double stranded DNA can permeate through the pore.
  • single stranded DNA may comprise an extension primer which has been extended
  • double stranded DNA may comprise an extension primer which has been extended, in addition to the complementary DNA strand.
  • one or more bases of the nucleic acid molecule is modified with a chemical tag.
  • the role of the chemical tag is to affect the ease with which the nucleic acid molecule may pass though the pore and therefore to alter the current signal.
  • current signal or “current signature” we refer to the specific alterations in both the duration and the amplitude of the current which result from the chemically modified nucleic acid molecule passing through the pore.
  • current signature can also include other characteristics such as current noise or characteristic time-dependent changes in the current blockades such as slopes of increasing or decreasing current with in a blockade event.
  • the size and type of the chemical tag is selected by consideration of the size of pore, and the degree to which it is desired to alter the ability of the nucleic acid molecule to a pass through the pore. Therefore the present invention is not limited in relation to the size or the type of the chemical tag. However, in some embodiments it may be preferred that the chemical modification is smaller than 1.5 nm diameter, more preferably smaller than 1 nm diameter, more preferably smaller than 0.5 nm diameter and most preferably about 0.3 nm diameter. The modification may act to increase the diameter of the nucleic acid molecule, thereby slowing its passage through the nanopore.
  • each type of chemical modification is associated with a specific base.
  • a single base may have one or more chemical modifications, but each chemical modification may be associated with only a single base and not two or more consecutive bases.
  • the primer is chemically modified with a chemical tag which allows identification, and optionally isolation, of the nucleic acid molecule (extended primer).
  • the pattern of chemical tags on a given primer is specific for the sequence of the primer and the pattern of chemical tags encodes for a specific and unique current signature which identifies the sequence of the primer and hence the sequence of the template the primer has bound to.
  • the base or bases which represent the SNP or mutation or are part of a highly repetitive sequence are chemically modified with one or more different chemical tags which have a different current signature. This allows detection and/or identification of the presence or concentration of the base.
  • the primer may contain more than four different types of tags.
  • tags For example, using solid phase oligonucleotide synthesis it is possible to generate an oligonucleotide which contains among other bases three adenines each of which carry a different chemical tags giving rise to a specific current signature. Given the multiple combinations of different chemical tags in the primer portion, it is possible to encode a multitude of different primers of different sequence. This enables multiplexing and the detection of several different SNPs or different mRNA types. The binding of the multiply labelled primers to the template is not affected by the presence of the chemical tags.
  • the chemical tags can be positioned at the portion of a primer which does not bind to the template strand.
  • the chemical tag is a peptide.
  • a modular tag in this case formed of amino acids, enables the skilled person to easily make numerous different tags with different current signatures.
  • the peptide tag consists of from 2 to 6 amino acids, but any suitable length may be used bearing in mind that the length of the peptide will correspond directly to the duration of the channel blockade and the duration component of the current signature.
  • suitable peptide tags are hexahistidine, hexaarginine, hexaaspartic acid, tetrahistidine, dihistidine and trityrosine.
  • Chemical tags that may be used in accordance with the invention include aromatic systems such as phenyl rings and substituted derivatives thereof, condensed aromatic systems such as naphthalene, phenanthrene, pyrene, and substituted derivatives thereof, saturated organic cyclic compounds such as cycloalkanes including cholesterol and derivatives thereof, spiro-molecule and other bicyclic compounds, tricyclic compounds such as adamantanes, monomeric and oligomeric carbohydrates such as cyclodextrins and substituted derivatives thereof, organometallic compounds including rhodium acetate complexes, metallocenes such as ferrocenes and substituted derivatives thereof, inorganic compounds, porphyrins and substituted organic derivatives thereof.
  • any number of chemical tags may be attached to the DNA molecule.
  • the number of chemical tags attached to the DNA may be altered for example to vary the ease with which molecules may pass through the pore and therefore the current pattern recorded.
  • a greater number of chemical tags will hinder the passage of molecules through the pore, whereas a smaller number will relatively ease the passage of molecules through the pore.
  • Embodiments of the invention are known in which one, two, three, four, five, six, seven, eight, nine or ten chemical tags are attached to the DNA molecule.
  • the chemical tag must be bound to the nucleotide in some way. The invention is not limited in respect of how this binding is achieved.
  • the chemical tag is attached to the nucleotide by various covalent linkages such as amide, disulfide, thioether bonds or linkages generated by Diels-Alder, Click-chemistry, or related pericyclic reactions.
  • the chemical linkages bind the chemical tags to the base.
  • purine bases carry a chemical linker at position 7 and pyrimidine bases at position 5. These positions are preferred because nucleotide analogues with linkers at these positions are known to be accepted as substrates by polymerase enzymes.
  • the chemical tags can be attached at various stages to the DNA.
  • synthetic oligonucleotides which are used as primers for the extension reaction can be obtained by linking the tag to a synthetic oligonucleotide carrying a modified base with a linker as shown in Example 1.
  • oligonucleotides with chemical tags can be obtained via solid phase oligonucleotides synthesis using phosphoramidite analogues carrying the chemical tags.
  • the chemical tags can be incorporated into a DNA strand by template-directed DNA polymerisation using triphosphate nucleotide analogues.
  • the polymerisation can be performed using nucleotide analogues carrying the chemical tags, or, in case the polymerase is not promiscuous, using analogues with linkers, which are derivatized with the chemical tag once the base has been incorporated into the DNA strand.
  • the tag When the tag is incorporated by polymerisation, either only the labelled nucleotide or a mixture of labelled nucleotide and unlabelled nucleotides are used, depending on the specific application. For example, if a primer should be extended by just one base, the labelled base, then the extension reaction is only performed in the presence of the labelled nucleotides (e.g. an 5-ethynyl-dUTP). If longer stretches should be extended, then more than one base can be included depending on the sequence. For the extension of the sequence UAG the bases 5-ethynyl-dUTP, dATP and dGTP would be used.
  • the labelled nucleotides e.g. an 5-ethynyl-dUTP
  • nucleotide mixture is, however, devoid of the unlabelled version of the labelled nucleotides.
  • a nucleotide mix may contains 5-ethynyl-dUTP but not dTTP.
  • primer extension is based on complementary base incorporation; misincorporations do not occur.
  • the different bases in the template direct the incorporation of one or more complementary labelled bases in the extended primer product.
  • each type of base may contain a specific chemical tag which gives rise to a specific current signature it is possible to infer which type of base is present in the extend primer strand by looking at the current signature.
  • Promiscuous polymerases such as Deep Vent exo- can be used for the incorporation of labelled nucleotides (JACS, 2006, 128, 1398-1399).
  • the primer may be designed to bind to the oligonucleotide template upstream (5') of the SNP, mutation or highly repetitive sequence.
  • the primer will bind to the template oligonucleotide less than 20, preferably less than 10 and most preferably 1 to 2 bases upstream of the SNP, mutation or highly repetitive sequence. This ensures that minimal extension of the primer is required in order to achieve labelled extension products.
  • the primer should preferably be designed to assist in interpretation of the current signals measured.
  • the SNP of interest is an adenine base and so labelled adenine nucleotides are to be incorporated into the extension product when the SNP is present, then it may be preferable to ensure that the primer is designed such that no other adenine residues are encoded prior to the SNP.
  • the current signal should be interpreted to take into account other labelled adenine residues prior to the one of interest.
  • the incorporation of chemically modified nucleotides can proceed in one step or in a two-step procedure.
  • the completely modified nucleotide is being incorporated into the DNA strands via polymerases.
  • Some of the artificial bulky nucleotides might be incorporated efficiently.
  • the problems of low incorporation yield can be overcome by a two-step procedure as shown in Fig. 6.
  • a nucleotide with a small linker is incorporated efficiently into DNA.
  • the remaining chemical tag is attached to the linker.
  • the remaining chemical tag can be attached to the base using a variety of chemistries such as Diels-Alder, Click-chemistry, or related pericyclic reactions, or disulfide exchange reactions.
  • the extended primer product may optionally be purified using the chemical tag in one of the extended bases or in the primer.
  • the extended primer product may contain a hexahistidine tag attached to a base in the extended section.
  • a histidine tag facilitates the purification of the modified DNA strand via immobilised metal affinity chromatography.
  • Other equally suitable methods of purification using chemical tags are well known to the person skilled in the art.
  • extended DNA will be purified from unextended primers and the biological templates because the latter do not contain histidine-tags.
  • the nanopore is the alpha-HL pore or another protein pore
  • only single stranded nucleic acid molecules can be translocated through because the narrow constriction of this pore is not wide enough for the passage of double stranded DNA.
  • the use of the single stranded DNA requires the isolation of the primer extension product and its separation from the unextended primer and the template sequence.
  • double stranded DNA could be used in combination with other nanopore such as inorganic pores. This would permit the analysis of an extended primer product which is still hybridized to the template sequence. It is, however, preferable to remove the template strand from the extended primer product due to the ill-defined length of the template. Should a long DNA or RNA strand cause the clogging of a nanopore, this may be cleared by the reversal of the transmembrane potential.
  • nucleic acid sample may be used in accordance with the invention.
  • sample may comprise numerous types of nucleic acid molecule and it will be apparent from the current measurements which are the molecules of interest.
  • nucleic acid molecules of interest can be purified prior to passing them through the nanopore, for example by using one of the chemical modifications to separate the nucleic acid molecules of interest using chromatography or the like as discussed above.
  • the method of the invention may be used for detecting single nucleotide polymorphisms (SNPs), for expression profiling, and for the sizing of highly repetitive sequences of forensic and biomedical importance.
  • SNPs single nucleotide polymorphisms
  • peptide tags can be incorporated into copied DNA strands from biological samples using chemically modified nucleotides and sequence-specific primer extension. This approach would be suitable to sense the presence or absence of single-nucleotide polymorphisms by incorporating and detecting a modified base only if the target mutation is present, or for sizing the highly repetitive DNA regions in trinucleotide expansion disease genes by labelling the same base in all repeats.
  • Fig. 4 The use of chemically modified DNA to detect SNPs using primer extension is illustrated in Fig. 4.
  • An oligonucleotide carrying two chemical tags binds to the template DNA strand.
  • the primer oligonucleotide carries two chemical tags which cause a specific current signature and therefore encode the identity of the primer.
  • Primer extension leads to the synthesis of the complementary DNA strand.
  • a chemically modified nucleotide is incorporated, while no chemical tag is added in the absence of the SNP in a different DNA strand (Fig. 4B).
  • the presence or absence of the SNP can be detected by monitoring the presence of absence of the additional blockade signature. This approach can be extended to analyse multiple different SNPs in a biological sample.
  • Multiple DNA primers may be modified with different sequences of chemical tags which give rise to characteristic patterns of current modulations.
  • the sequence of the primer can therefore be inferred from the characteristic pattern of current modulations in the traces. Detecting the signatures in the sample with the extended primer therefore indicates that a specific SNP must have been present in the original sample.
  • Fig. 5 The use of chemically modified DNA to size the length of highly repetitive DNA strands is illustrated in Fig. 5.
  • the expression "highly repetitive sequence” is well known in the art. Repetitive DNA sequences with a length range from a hundred to a few thousand bases are found in non-coding regions of the human genome in the form of microsatellite DNA. These sequences are composed of terra-, tri-, or dinucleotides such as CA repeats and recur 10 to 100 times without interruption. As the number of repeats at a given chromosomal locus is characteristic for each individual, microsatellite DNA is used for genotyping in recombination mapping, studies on population genetic, and paternity tests.
  • Prominent examples of repeat sequences occurring in coding genome regions are genes of trinucleotide expansion diseases such as Huntington's, Myotonic dystrophy and Friedreich's ataxia.
  • the expanded trinucleotide regions arise due to strand slippage during DNA replication and have been identified as molecular reason for pathogenic changes (Bates, G. P. (2005) Nat Rev Genet 6, 766-73, Li, S. H., and Li, X. J. (2004) Trends Genet 20, 146-54).
  • expanded CAG repeats encode for polyglutamate tracts, which can lead to misfolding, aggregation and dysfunction of the disease-relevant proteins (Yoon, S.
  • Fig. 5A in the case of the presence of two repeats of a highly repetitive sequence, two chemically modified nucleotides are incorporated resulting in an additional blockade signature of length 2xt 2 .
  • Fig. 5B shows that in the case of the presence of seven repeats, seven chemically modified nucleotides are incorporated resulting in an additional blockade signature of length 7xt 2 . In this way it is possible to distinguish between the numbers of repeats of the sequence of interest.
  • the proportions of two or more types of mRNA from one or two different samples may be identified to obtain information about the physiological or disease state of cells and tissues. For example mRNA from different genes may be compared or mRNA from different cell types or organisms may be compared.
  • the relative proportion of two different types of mRNA within one tissue sample is analysed.
  • Two different primers are used, which recognise specifically bind to either one of the mRNA templates.
  • the primers differ not only in their base sequence but also in the pattern of the chemical tags such as arginine or tyrosine (but preferably not histidine) peptides which encode for a specific and distinguishable current signature.
  • the primers are extended by at least one additional nucleotide.
  • the extended nucleotide carries a chemical tag such as the ethynyl group which is then reacted with the azido-derivatized histidine peptide in a Click-reaction as shown in example 3 and 4.
  • the primer extension products may be purified using for example immobilised metal affinity chromatography as illustrated in example 1.
  • the two extended primers are analysed using single channel current recordings.
  • the translocation of the two DNA strands gives rise to two different current signatures.
  • the current signatures are composed of two segments; the first is caused by the primer-specific tags and the other by the extension specific tag.
  • the extension specific tag and signature is identical for both types of DNA but the primer-specific tags are different and allow us to discriminate between the two different extended primer products. It should be noted that the signature caused by the extension specific tag is not absolutely required as the histidine tag has already been used to separate unextended from extended primers. The presence of the extension signature is nevertheless an additional criterion to positively identify the extended primer.
  • the current recordings may be performed to sense at least 1000 events for the two types of extended primer products.
  • the proportion of the events is proportional to the ratio of the two mRNA types in the biological sample. Additional calibration with reference standards of known ratios of extended primer products can be conducted as an option to enhance the accuracy of the measurements.
  • nanopore recordings use electrical rather than optical signals to sense biomolecules.
  • a low-cost miniaturized electrical read-out device which carries out the method according to the invention could be used in point-of-care applications.
  • 3-(2-Pyridyldithio)-propanoic acid was synthesized as a precursor for the generation of ⁇ /-succinimidyl 3-(2-pyridyldithio)-propanoate (SPDP).
  • SPDP ⁇ /-succinimidyl 3-(2-pyridyldithio)-propanoate
  • DPDS 2,2'-Dipyridyldisulfide
  • Acetic acid 1.5 mL
  • 3-mercapto-propanoic acid (2.64 g, 2.16 ml, 0.0249 mol) were added and the solution left stirring at room temperature for 2 hours.
  • CHHHHHH peptide was synthesised using standard automated fmoc SPPS chemistry on a Syro automated peptide synthesiser using HBTU coupling chemistry
  • the peptide was further purified via RP-HPLC - 5 mg was dissolved in 300 ⁇ L of 0.1% TFA water. 50 - 100 ⁇ L aliquots were purified per run using a 5 ml/min semi prep column; gradient 2 - 10% B over 15min (A - 0.1% TFA water, B 0.1% TFA acetonitrile). The fractions containing the correct peak were pooled and freeze dried overnight to leave a white powder - single peak on analytical HPLC. Calculated mass ES + ESI: (m/z) - 473.22 (MH 2+ ), 944.99 (MH + ), 966.91 (M + Na + ).
  • SPDP N-Succinimidyl 3-(2-pyridyldithio)-propanoate
  • the sample was agitated at room temp for 2 hrs before the volume was reduced on the vac. centrifuge to approx. 500 ⁇ L.
  • the POC was purified via anion-exchange chromatography (1 ml/min HiTrapp Q FF col.) using a linear gradient (0 - 40% B) over 90 CV (buffer A - 20 mM Tris, pH 8.0; buffer B - 20 mM Tris, 2M NaCl, pH 8.0). The fractions containing the product (elution time 10 ml) were pool into 2 ml and desalted using 4 mL (5KDa MW cut-off) desalting columns.
  • 2', 4', 6'- Trihydroxyacetophenone (THAP) matrix 25 mg was weighed out and dissolved in 250 ⁇ L of methanol.
  • Imidazole 0.2 g was dissolved in 10 ml of water and vortexed until dissolved.
  • the oligonucleotide pellet was dissolved in 50 ⁇ L of nano- pure water and 2 ⁇ L of the matrix solution was mixed well with 2 ⁇ L of the oligonucleotide sample and this solution was then spotted onto the MALDI plate.
  • 1 ⁇ L of co-matrix in this case imidazole was added to the droplet and the sample was allowed to crystallize before being analysed via MALDI MS.
  • the POC was also analysed via Ni 2+ affinity columns on the AKTA system (step gradient from 0% B 10 column volumes (CV) to 100% B lOCV using the following buffers: A - 0.1 M Na 2 HPO 4 , 50 mM NaCl, pH 8.0; B - 0.1 M Na 2 HPO 4 , 50 mM NaCl, IM Imidazole, pH 8.0).
  • the Histidine peptide-DNA conjugate eluted with the imidazole-containing buffer.
  • the procedure detail above was used to modify two 27mer oligonucleotides with internal amine modifications with CG 6 and CH 6 peptides respectively.
  • the second POC also contained a 5 ' phosphate group to allow ligation:
  • the following buffer was made up to 10 mL; 50 mM Tris. HCl, 10 mM MgCl 2 , mM ATP, pH 7.5 and cooled to 16 0 C. To 370.4 ⁇ L of this solution the hybridized DNA solution was added followed by the T4 solution, and left overnight (0.025 mM). The sample was diluted up to 1 mL in the following buffer A and run through an anion - exchange col. using a gradient of 0 - 40% B over 90 CV in buffers; A - 2OmM Tris, 8M Urea, pH 8.0; B - 2OmM Tris, 2M NaCl, 8M Urea, pH 8.0.
  • the short linking oligo eluted at 16 ml while the longer DNA strand with two chemical tags eluted at 21 ml.
  • the solution was then desalted using 4 mL, 5KDa MW cutt-off desalting column and prepared for MALDI by precipitating in 2.5 vol absolute ethanol in the presence of 1/3 vol. 1OM ammonium acetate.
  • a broad M 1" peak at 18232.5 m/z was observed (18236 m/z expected) along with a sharp M 3" peak at 6181.7 m/z (6075.66 m/z expected).
  • a 15% TBE polyacrylamide gel was also run confirming that the gel shift of the double- modified DNA strand.
  • 5-iodo-2'-deoxyuridine 370 mg, 1.04 mmol
  • palladium tetrakis triphenylphosphine 113mg, 0.098 mmol
  • copper iodide 47mg, 0.25mmol
  • Anhydrous DMF (12.0 mL) was added via syringe, followed by triethylamine (0.4 mL, 2.9 mmol) and trimethylsilylacetylene (0.7ImL, 5.0 mmol).
  • 5-ethynyl-2'-deoxyuridine (12 mg, 0.05 mmol) and azido-acetic acid (6mg, 0.06 mmol) were dissolved in a NaH 2 PO 4 buffer (pH 6.2, 0.12M, 1 mL) and heated at 37°C to ensure dissolution of the reagents. Then a freshly made sodium ascorbate aqueous solution (25 ⁇ L, IM) and a freshly made CuSO4 aqueous solution (25 ⁇ L, 0.1M) were added and the reaction mixture was maintained at 37°C for 130min, at which point, analytical HPLC of a sample of the solution indicated complete consumption of the starting material nucleoside. The mixture was filtered through microfilter (minisart 0.20 ⁇ M) and the filtrates were purified by HPLC.
  • PCR amplification of 500 bp fragments were carried out using 50 ⁇ L reaction mixtures each containing 10x thermopol buffer (NEB) (5 ⁇ L), lOO ⁇ M Primer forward 5150 (2 ⁇ L) (5'-CCAACA GGT GCAAAT GTT TAC GGT C), lOO ⁇ M primer reverse 5578 (2 ⁇ L) (5'-ATG CTA GTT ATT GCT CAG CGG TGG), 25 nM SbsB T433C template (J. Duranton, C. Boudier, D. Belorgey, P. Mellet, J.
  • PCR amplification was performed using a thermocycler with the following conditions: step 1 (2 min at 94°C); 30 cycles of step 2 (1 min at 95°C, 1 min at 54°C, 1 min at 72°C), step 3 (10 min at 72°C).
  • step 1 (2 min at 94°C); 30 cycles of step 2 (1 min at 95°C, 1 min at 54°C, 1 min at 72°C), step 3 (10 min at 72°C).
  • the three DNA samples were analysed by polyacrylamide gel electrophoresis (10%) and ethidium bromide staining. Samples 1 and 3 displayed a DNA band at 500 bp, sample 2 did not show any DNA band.
  • the Click reaction was carried following a published procedure (Org. Lett. 2006, 8, 3639-3642) by mixing PCR mix 1 and 3 (45 ⁇ L) with 1 mM CuSO 4 solution (5 ⁇ L), 10 mM ascorbate solution (5 ⁇ L), and 20 mM peptide azido-GGGHHHH (4.5 ⁇ L). The mixtures were incubated at 37°C for 90 min, and analysed by gel electrophoresis (10% polyacrylamide gel). Sample from the ethynyl-base containing sample showed an upshifted band compared to the band from the sample without ethynyl groups.
  • Each compartment contained 1.0 ml of 2 M KCl, 20 mM Tris ⁇ Cl pH 7.5.
  • Gel-purified heptameric ⁇ HL protein (final concentration 0.01-0.1 ng/ml) was added to the cis compartment, and the electrolyte in the cis chamber was stirred until a single channel inserted into the bilayer.
  • Transmembrane currents were recorded at a holding potential of +100 mV (with the cis side grounded) by using a patch-clamp amplifier (Axopatch 200B, Axon Instruments, Union City, CA).
  • FIG. 7A A typical current single-channel current trace with events caused by hexa-arginine modified oligonucleotide of 27 bases in length is shown in Fig. 7A.
  • Analysis of more than 400 events yielded a characteristic current blockade of more than 205 pA which constitutes more than 99% of the open channel current.
  • the average duration of the events was obtained form the fitting of the dwell-time distribution with a single exponential and yielded a value of 12 ms.
  • Fig. 7B events of the DNA strand carrying a hexa-histidine tag (Fig. 7B) were characterised by a current blockade of 94% and an average duration of 1.84 ms.
  • the DNA oligonucleotide without any peptide tag had an average duration of 0.14 ms and a current blockade of 80% (Fig. 7C).
  • the recordings demonstrate that a chemical tag such as a short oligopeptide slows down the passage of a DNA strand through the pore.
  • tags of the same length but different chemical composition such as hexaarginine and hexahistidine led to different current signatures.
  • blockade duration can be varied by changing the length of the peptide.
  • Three peptides with 2, 4 or 6 histidines were tested and the blockade characteristics for 500 events each are summarized in the table. Within this histidine peptide series, the blockade durations correlated directly with the length of the peptides.
  • DNA strands carrying two peptide tags one with a hexa-histidine the other with a tri-tyrosine tag from example 2 was analysed with single channel current recordings. A typical event is shown in Fig. 7D.
  • primer extension is used to detect single nucleotide polymorphisms in biomedically relevant sequences.
  • a single point mutation in the HIV-2 gene encoding for the protease is detected.
  • the first point mutation at codon position 90 from TTG -> ATG encodes leads to an amino acid change from leucine (L) to methionine (M).
  • the section of the HIV-2 sequence around position 90 of the wild type is: 5'-AT TGG AAG AAA TCT GTT GAC TCA GAT TGG TTG CAC TTT AA-3' and for the drug resistance conferring mutation is
  • the extended bases are indicated in bold.
  • the U base carries the ethynyl group.
  • the sequence around position 23 is 5 ' AAA GGA AGC TCT ATT AGA TAC AGG AGC AGA TGA TAC AGT while the sequence for the single-point mutant is 5' AAA GGA AGC TAT ATT AGA TAC AGG AGC AGA TGA TAC AGT.
  • primer extension product Performing a primer extension using a primer with the sequence 5 ' ACT GTA TCA TCT GCT CCT GTA TCT AAT A-3 ' and nucleotides dATP, dGTP, and ethynyl-dUTP, the following primer extension product are formed:
  • the ethynl containing uridine base in the extended products is modified with His- tagged peptide as described in example 3 and 4, and the tagged extended primers products are purified as described in example 1.
  • the histidine-tag causes a specific current signature in the recordings.
  • the primer sequence itself contains additional peptide sequences and gives rise to characteristic current signatures as described in example 5, and allows us to distinguish between the primers for position 90 and 23.
  • the short events represent the fast translocation of individual strands from the cis to the trans side of the pore ( Figure 8D) (Kasianowicz et al, Proc. Nat. Acad. Sci. U S A 1996, 93, 13770; Butler et al, Biophys. J. 2006, 90, 190).
  • the recordings also displayed blockades with 50% amplitude, which were not pursued further as they likely represent the reversible threading of a strand into and the escape from the cis opening rather than the complete translocation to the trans side.
  • Type I events ( Figure 8E) had a high-amplitude blockade, Ah of 96.8 ⁇ 0.5 % with an average duration, ⁇ o ff-h, of 1.83 ⁇ 0.26 ms. Due to the this very defined blockade, type I events certainly represent complete pore translocation. By comparison, type II events ( Figure 8E) started with a mid-amplitude level, A m , of 56.6 ⁇ 2.6 % with a duration, ⁇ o ff -m , of 1.34 ⁇ 0.36 ms.
  • Ol carrying a H 6 Ci tag at a terminal rather than an internal position did not greatly retard DNA passage as shown by a short event time of 0.23 ⁇ 0.10 ms ( Figure lF)(Table 1, H 6 C-Ol -term.). The absence of a major retardation is attributed to the fact that the peptide can sequentially pass the pore after the DNA strand without the formation of a bulky peptide-DNA segment.
  • the peptides tags are certainly the molecular reason of the retardation and may exert their effect by either hindered diffusion or an increase in friction mediated by steric, electrostatic, polar, and/or hydrophobic interactions. (Mathe et al, Proc. Nat. Acad. ScL USA 2005, 102, 12377; Kathawalla et al, Macromolecules 1989, 22, 1215).
  • step-like blocking effect of Y 3 Ci was independent of the DNA sequence around the modified base because the same event characteristics were also seen for Y 3 Ci-O2 with a different sequence (Table 1; Y 3 Ci-Ol /step vs Y 3 Ci-O2 /step).
  • the first strand was a 37-mer Y 3 /Y 3 -O3 in which two Y 3 Ci peptides are tethered to two modified bases separated by 13 nucleotides. Similar to single modified Y 3 Ci-Ol strand, double modified DNA gave rise to unresolved slope events (Supporting Information) as well as fully resolved step-like events (Figure 9D). In the latter events, the blockade amplitude fluctuates twice between two levels sequentially from event segments 1 to 4 ( Figure 9D, event segments numbered red). The average current levels for segments 1 and 3, and 2 and 4 are 92 and 99%, respectively (Table 2).
  • the step-like signature is in line with expectations for two Y 3 Ci peptides because one peptide is known to cause a blockade step from 92 % to 98 % (Table 1, Y 3 Ci-I /step).
  • the signature of Y 3 /Y 3 -O3 in Figure 9D strongly suggests that the current alterations reflect the sequential pulling of a DNA strand through the pore as illustrated schematically in Figure 9F (numbers correspond to segments in Figure 2D).
  • Table 2 Characteristics of type I translocation events of Y3/Y3-O3 and Y3/Y3-O4 carrying tags separated by 13 and 27 nt, respectively ⁇

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Abstract

L'invention concerne un procédé de détection d'acides nucléiques. Selon l'invention, l'utilisation d'enregistrements de courant à canal unique (nanopore) conjointement avec de l'ADN chimiquement modifié pour récupérer des informations spécifiques à la séquence peut être appliquée à la détection de polymorphismes d'un seul nucléotide (SNP), au profilage d'expression et au calibrage de séquences hautement répétitives d'importance biomédicale et médico-légale.
PCT/GB2008/050536 2007-07-06 2008-07-04 Procédé de détection d'acides nucléiques WO2009007743A1 (fr)

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