Electrochemical sensor using intercalative, redox-active moieties
First Claim
1. A method of detecting one or more single-base sequence lesions in a target nucleic acid sequence, comprising:
- a) contacting a first single stranded nucleic acid probe sequence with a second single stranded nucleic acid target sequence to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded nucleic acid probe sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid probe sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid probe sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;
b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety and a non-intercalative redox-active moiety; and
c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge from the electrical current or charge of a nucleic acid duplex not having one or more single-base sequence lesions is indicative of the presence or absence of one or more single-base sequence lesions in the target sequence.
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Accused Products
Abstract
Compositions and methods for electrochemical detection and localization of genetic point mutations, common DNA lesions and other base-stacking perturbations within oligonucleotide duplexes adsorbed onto electrodes and their use in biosensing technologies are described. An intercalative, redox-active moiety (such as an intercalator or nucleic acid-binding protein) is adhered and/or crosslinked to immobilized DNA duplexes at different separations from an electrode and probed electrochemically in the presence or absence of a non-intercalative, redox-active moiety. Interruptions in DNA-mediated electron-transfer caused by base-stacking perturbations, such as mutations or binding of a protein to its recognition site are reflected in a difference in electrical current, charge and/or potential.
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Citations
100 Claims
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1. A method of detecting one or more single-base sequence lesions in a target nucleic acid sequence, comprising:
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a) contacting a first single stranded nucleic acid probe sequence with a second single stranded nucleic acid target sequence to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded nucleic acid probe sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid probe sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid probe sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;
b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety and a non-intercalative redox-active moiety; and
c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge from the electrical current or charge of a nucleic acid duplex not having one or more single-base sequence lesions is indicative of the presence or absence of one or more single-base sequence lesions in the target sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A method of detecting one or more single-base sequence lesions in a target nucleic acid sequence, comprising:
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a) contacting a first single stranded, derivatized nucleic acid probe sequence with a second single stranded nucleic acid target sequence to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded derivatized nucleic acid probe sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded, derivatized nucleic acid probe sequence to the electrode, and dehybridizing the duplex, such that the first single stranded, derivatized nucleic acid probe sequence remains attached to the electrode, and wherein the hybrid of the first single stranded, derivatized nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;
b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety and a non-intercalative redox-active moiety; and
c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge to the electrical current or charge of a nucleic acid duplex not having one or more single-base sequence lesions is indicative of the presence or absence of one or more single-base sequence lesions in the target sequence. - View Dependent Claims (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 30, 31, 32, 33, 34, 35, 37, 38, 39, 40, 41, 42)
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23. A method of detecting one or more single-base sequence lesions in a target nucleic acid sequence, comprising:
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a) contacting a first single stranded nucleic acid probe sequence with a second single stranded nucleic acid target sequence to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded nucleic acid probe sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid probe sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid probe sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;
b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety, wherein the intercalative, redox-active moiety is intercalatively stacked and a non-intercalative redox-active moiety; and
c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge to the electrical current or charge of a nucleic acid duplex not having one or more single-base sequence lesions is indicative of the presence or absence of one or more single-base sequence lesions in the target sequence.
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29. A method of detecting one or more single-base mismatches in a target nucleic acid sequence, comprising:
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a) contacting a first single stranded, derivatized nucleic acid probe sequence with a second single stranded nucleic acid target sequence to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded derivatized nucleic acid probe sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid probe sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid probe sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;
b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety and a non-intercalative redox-active moiety; and
c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge to the electrical current or charge of a nucleic acid duplex not having one or more single-base sequence mismatches is indicative of the presence or absence of one or more single-base sequence mismatches in the target sequence.
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36. A method of detecting one or more single-base sequence lesions in a portion of a gene sequence, wherein the one or more single-base sequence lesions is associated with a disease, comprising:
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a) contacting a first single stranded nucleic acid probe sequence, comprising a derivatized portion of the sequence of a gene, with a second single stranded nucleic acid target sequence comprising a wild type portion of the gene, to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded nucleic acid probe sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid probe sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid probe sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;
b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety and a non-intercalative redox-active moiety; and
c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge to the electrical current or charge of a nucleic acid duplex not having one or more single-base sequence lesions is indicative of the presence or absence of one or more single-base sequence lesions in the gene sequence wherein the base stacking perturbation or single-base sequence lesion is associated with a disease.
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43. A method of detecting one or more naturally occurring DNA lesions in a target nucleic acid sequence, comprising:
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a) contacting a first single stranded nucleic acid probe sequence containing a DNA lesion with a second single stranded nucleic acid target sequence to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded nucleic acid probe sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid probe sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid probe sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;
b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety and a non-intercalative redox-active moiety; and
c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge to the electrical current or charge of a nucleic acid duplex not having one or more DNA lesions is indicative of the presence or absence of one or more DNA lesions in the target sequence. - View Dependent Claims (44, 45, 46, 47, 48, 49, 50, 51, 52, 53)
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54. A method of detecting one or more single-base sequence lesions in a target nucleic acid sequence, comprising:
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a) contacting a first single stranded nucleic acid probe sequence with a second single stranded nucleic acid target sequence to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded nucleic acid probe sequences on an addressable multielectrode array, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid probe sequence to the addressable multielectrode array, and dehybridizing the duplex, such that the first single stranded nucleic acid probe sequence remains attached to the addressable multielectrode array, and wherein the hybrid of the first single stranded nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;
b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety and a non-intercalative redox-active moiety; and
c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge from the electrical current or charge of a nucleic acid duplex not having one or more single-base sequence lesions is indicative of the presence or absence of one or more single-base sequence lesions in the target sequence. - View Dependent Claims (55, 56, 57, 58, 59)
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60. A method of detecting one or more single-base sequence lesions in a target nucleic acid sequence, comprising:
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a) contacting a first single stranded nucleic acid probe sequence with a second single stranded nucleic acid target sequence to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded nucleic acid probe sequences on an ultramicroelectrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid probe sequence to the ultramicroelectrode, and dehybridizing the duplex, such that the first single stranded nucleic acid probe sequence remains attached to the ultramicroelectrode, and wherein the hybrid of the first single stranded nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;
b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety and a non-intercalative redox-active moiety; and
c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge from the electrical current or charge of a nucleic acid duplex not having one or more single-base sequence lesions is indicative of the presence or absence of one or more single-base sequence lesions in the target sequence. - View Dependent Claims (61, 62)
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63. A method of detecting one or more single-base sequence lesions in a hybrid of DNA and RNA, comprising:
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a) contacting a first single stranded nucleic acid probe sequence with a second single stranded nucleic acid target sequence to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded nucleic acid probe sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid probe sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid probe sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;
b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety and a non-intercalative redox-active moiety; and
c) measuring the electrical current or charges of the catalytic reduction of the non- inter(alative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge to the electrical current or charge of a nucleic acid duplex not having one or more single-base sequence lesions is indicative of the presence or absence of the one or more single-base sequence lesions in a hybrid of DNA and RNA. - View Dependent Claims (64, 65, 66, 68, 69)
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67. A method of detecting one or more single-base sequence lesions in a portion of a derivatized wild type target nucleic acid sample, comprising:
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a) contacting a first single stranded nucleic acid probe sequence with a second single stranded nucleic acid target sequence, comprising a portion of a derivatized wild type target nucleic acid sample, to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded nucleic acid probe sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid probe sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid probe sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;
b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety and a non-intercalative redox-active moiety; and
c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge to the electrical current or charge of a nucleic acid duplex not having one or more single-base sequence lesions is indicative of the presence or absence of one or more single-base sequence lesions in a portion of the derivatized wild type target nucleic acid sample.
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70. A method of detecting base flipping in a duplex nucleic acid sequence associated with protein binding to the duplex nucleic acid sequence, comprising:
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a) contacting a first single stranded derivatized nucleic acid sequence, wherein the first single stranded nucleic acid sequence contains a protein binding sequence and an electrochemical probe binding sequence, with a second single stranded nucleic acid sequence to form a duplex, wherein the second single stranded nucleic acid sequence is hybridized to a monolayer of first single stranded nucleic acid sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid sequence and the second single stranded sequence form a double stranded nucleic acid-modified film;
b) adding an electrochemical probe to the double stranded nucleic acid-modified film, wherein the electrochemical probe binds to the electrochemical probe binding sequence of the first single stranded nucleic acid sequence;
c) adding a protein to the double stranded nucleic acid modified film, wherein the protein binds to the protein binding sequence of the first single stranded nucleic acid sequence; and
d) measuring the electrical current or charges of the reduction of the electrochemical probe, wherein a difference of the electrical current of charge from the electrical current or charge of a nucleic acid duplex not having a protein bound to the duplex DNA is indicative of the presence or absence of base flipping due to protein binding in the duplex. - View Dependent Claims (71, 72, 73, 74, 75, 76, 77, 78, 79)
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80. A method of detecting base stacking perturbations in a duplex nucleic acid sequence associated with protein binding to the duplex nucleic acid sequence, comprising:
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a) contacting a first single stranded derivatized nucleic acid sequence, wherein the first single stranded nucleic acid sequence contains a protein binding sequence and an electrochemical probe binding sequence, with a second single stranded nucleic acid sequence to form a duplex, wherein the second single stranded nucleic acid sequence is hybridized to a monolayer of first single stranded nucleic acid sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid sequence and the second single stranded sequence form a double stranded nucleic acid-modified film;
b) adding an electrochemical probe to the double stranded nucleic acid-modified film, wherein the electrochemical probe binds to the electrochemical probe binding sequence of the first single stranded nucleic acid sequence;
c) adding a protein to the double stranded nucleic acid modified film, wherein the protein binds to the protein binding sequence of the first single stranded nucleic acid sequence; and
d) measuring the electrical current or charges of the reduction of the electrochemical probe, wherein a difference of the electrical current of charge from the electrical current or charge of a nucleic acid duplex not having a protein bound to the duplex DNA is indicative of the presence or absence of one or more base stacking perturbations in the duplex. - View Dependent Claims (81, 82, 83, 84, 85, 86, 87, 88, 89)
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90. A method of electrical monitoring of DNA enzymatic reactions in a duplex nucleic acid sequence associated with protein binding to the duplex nucleic acid sequence, comprising:
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a) contacting a first single stranded derivatized nucleic acid sequence, wherein the first single stranded nucleic acid sequence contains a protein binding sequence and an electrochemical probe binding sequence, with a second single stranded nucleic acid sequence to form a duplex, wherein the second single stranded nucleic acid sequence is hybridized to a monolayer of first single stranded nucleic acid sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid sequence and the second single stranded sequence form a double stranded nucleic acid-modified film;
b) adding an electrochemical probe to the double stranded nucleic acid-modified film, wherein the electrochemical probe binds to the electrochemical probe binding sequence of the first single stranded nucleic acid sequence;
c) adding a protein to the double stranded nucleic acid modified film, wherein the protein binds to the protein binding sequence of the first single stranded nucleic acid sequence; and
d) measuring the electrical current or charges of the reduction of the electrochemical probe, wherein a difference of the electrical current of charge from the electrical current or charge of a nucleic acid duplex not having a protein bound to the duplex DNA is indicative of the presence or absence of an enzymatic reaction of the duplex. - View Dependent Claims (91, 92, 93, 94, 95, 96, 97, 98, 99, 100)
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Specification