Polymer tagged nucleotides for single molecule electronic snp assay

Abstract

This invention provides methods of using labeled nucleotide polyphosphate analogues to detect the identity or presence of a nucleotide at certain positions in nucleic acid sequences with single molecule sensitivity using nanopore detection, nucleotides and primer-conjugated nanopore proteins for use in such methods, and processes for producing such nucleotides and primer-conjugated nanopore proteins.

What is claimed is: 
     
         1 . A method for identifying a single nucleotide residue of interest at a position within a stretch of consecutive nucleotide residues in a nucleic acid, comprising the steps of:
 a) incubating the nucleic acid, under an applied voltage, with
 (1) a nanopore; 
 (2) an oligonucleotide primer conjugated to the nanopore and hybridized to the nucleotides in the nucleic acid immediately 3′ to the single nucleotide residue of interest; 
 (3) at least one labeled terminating nucleotide polyphosphate (NPP) analogue, wherein the label is attached to either the base or the terminal phosphate of the NPP analogue; and 
 (4) a nucleic acid polymerase; 
 so that a NPP analogue is incorporated into the primer if it is complementary to the single nucleotide residue of interest, and the label attached to the incorporated NPP analogue is drawn into the nanopore; 
   b) detecting by nanopore the signature of the label of the NPP analogue incorporated into the primer, so as to identify the incorporated NPP analogue;   thereby identifying the single nucleotide residue of interest.   
     
     
         2 . The method of  claim 1 , wherein in step (a) the nucleic acid is incubated with at least two NPP analogues, each comprising a different base or analogue of a base, and a distinct label. 
     
     
         3 . The method of  claim 2 , wherein in step (a) the nucleic acid is incubated with at least four NPP analogues, each comprising a different base or analogue of a base, and a distinct label. 
     
     
         4 . The method of  claim 3 , wherein in step (a) the nucleic acid is incubated with exactly four NPP analogues, each comprising a different base or analogue of a base, and a distinct label. 
     
     
         5 . The method of  claim 1 , wherein in step (a) the nucleic acid is incubated with one NPP analogue, and if the NPP analogue is not incorporated, iteratively repeating the incubating with a different NPP analogue until a NPP analogue is incorporated and its label detected by nanopore. 
     
     
         6 . A method for identifying a single nucleotide residue of interest at a position within a stretch of consecutive nucleotide residues in a nucleic acid, comprising the steps of:
 (a) incubating the nucleic acid, under an applied voltage, with
 (1) a nanopore; 
 (2) an oligonucleotide primer conjugated to the nanopore and hybridized to the nucleotides in the nucleic acid immediately 3′ to the single nucleotide of interest; 
 (3) at least one labeled nucleotide polyphosphate (NPP) analogue, wherein the label is attached to either the base or the terminal phosphate of the NPP analogue; 
 (4) a nucleic acid polymerase; and 
 (5) a non-catalytic ion which permits transient binding of a complementary labeled NPP analogue to the nucleic acid polymerase but inhibits incorporation of the bound NPP analogue; 
 so that a NPP analogue is transiently bound to the nucleic acid polymerase if it is complementary to the single nucleotide of interest, and the label attached to the transiently bound NPP analogue is drawn into the nanopore; 
   (b) detecting by nanopore the signature of the label of the NPP analogue transiently bound to the primed template by polymerase, so as to identify the NPP analogue;   thereby identifying the single nucleotide residue of interest.   
     
     
         7 . The method of  claim 6 , wherein in step (a) the nucleic acid is incubated with at least two NPP analogues, each comprising a different base or analogue of a base, and a distinct label. 
     
     
         8 . The method of  claim 7 , wherein in step (a) the nucleic acid is incubated with at least four NPP analogues, each comprising a different base or analogue of a base, and a distinct label. 
     
     
         9 . The method of  claim 8 , wherein in step (a) the nucleic acid is incubated with exactly four NPP analogues, each comprising a different base or analogue of a base, and a distinct label. 
     
     
         10 . The method of  claim 6 , wherein in step (a) the nucleic acid is incubated with one NPP analogue, and if the NPP analogue does not transiently bind to the nucleic acid polymerase, iteratively repeating the incubating with a different NPP analogue until a NPP analogue is transiently bound and its label detected by nanopore. 
     
     
         11 . The method of any one of  claims 6 - 10 , wherein the non-catalytic metal ion is Sr 2+  or Ca 2+ . 
     
     
         12 . The method of any one of  claims 6 - 11 , wherein each NPP is a deoxyribonucleotide polyphosphate (dNPP). 
     
     
         13 . The method of any one of  claims 1 - 11 , wherein each NPP is a dideoxyribonucleotide polyphosphate (ddNPP). 
     
     
         14 . The method of  claim 13 , wherein each ddNPP comprises a label having a coumarin-PEG moiety. 
     
     
         15 . The method of  claim 13 , wherein each ddNPP comprises a label having an oligonucleotide-based tag. 
     
     
         16 . The method of any one of  claims 6 - 11 , wherein each NPP is a ribonucleotide polyphosphate (rNPP). 
     
     
         17 . The method of any one of  claims 1 - 16 , wherein the nucleic acid is single-stranded DNA. 
     
     
         18 . The method of any one of  claims 1 - 16 , wherein the nucleic acid is double-stranded DNA. 
     
     
         19 . The method of any one of  claims 1 - 16 , wherein the nucleic acid is single-stranded RNA. 
     
     
         20 . The method of any one of  claims 1 - 16 , wherein the nucleic acid is double-stranded RNA. 
     
     
         21 . The method of any one of  claims 12 - 13 , wherein the nucleic acid is single-stranded DNA or double-stranded DNA and the nucleic acid polymerase is DNA polymerase. 
     
     
         22 . The method of any one of  claims 12 - 13 , wherein the nucleic acid is RNA and the nucleic acid polymerase is reverse transcriptase. 
     
     
         23 . The method of any one of  claims 12 - 13  and  16 , wherein the nucleic acid polymerase is RNA polymerase. 
     
     
         24 . The method of any one of  claims 1 - 23 , wherein the label is attached to the base. 
     
     
         25 . The method of any one of  claims 1 - 23 , wherein the label is attached to the terminal phosphate. 
     
     
         26 . The method of any one of  claims 1 - 25 , wherein the label comprises one or more of ethylene glycol, an amino acid, a carbohydrate, a peptide, a dye, a chemiluminescent compound, a mononucleotide, a dinucleotide, a trinucleotide, a tetranucleotide, a pentanucleotide, a hexanucleotide, an oligonucleotide, an aliphatic acid, an aromatic acid, an alcohol, a thiol group, a cyano group, a nitro group, an alkyl group, an alkenyl group, an alkynyl group, an azido group, or a combination thereof. 
     
     
         27 . The method of any one of  claims 1 - 26 , wherein the label is a polymeric label. 
     
     
         28 . The method of any one of  claims 1 - 27 , wherein the labels are polyethylene glycol (PEG) labels. 
     
     
         29 . The method of  claim 28 , wherein the PEG labels each have a different length from each other. 
     
     
         30 . The method of any one of  claims 1 - 29 , wherein the labels are oligonucleotide labels. 
     
     
         31 . The method of any one of  claims 1 - 30 , wherein the signature is an electronic signature. 
     
     
         32 . The method of  claim 31 , wherein the electronic signature is an electrical current blockade signature. 
     
     
         33 . The method of  claim 32 , wherein the electrical current blockade signature is a stuttering current blockade signature. 
     
     
         34 . The method of any one of  claims 1 - 33 , wherein the nanopore is a solid-state nanopore. 
     
     
         35 . The method of any one of  claims 1 - 33 , wherein the nanopore is in a solid state membrane. 
     
     
         36 . The method of any one of  claims 1 - 33 , wherein the nanopore is a biological pore. 
     
     
         37 . The method of any one of  claims 1 - 33 , wherein the nanopore is proteinaceous. 
     
     
         38 . The method of  claim 37 , wherein the nanopore comprises alpha hemolysin. 
     
     
         39 . The method of  claim 38 , wherein each nanopore comprises seven alpha hemolysin monomers, any or all of which are conjugated to an identical primer. 
     
     
         40 . The method of  claim 38 , wherein each nanopore comprises eight MspA monomers, any or all of which are conjugated to an identical primer. 
     
     
         41 . The method of  claim 38 , wherein each nanopore comprises nine CsgG monomers, any or all of which are conjugated to an identical primer. 
     
     
         42 . The method of  claim 14 , wherein the nucleic acid is incubated with four coumarin-PEG-ddNPPs, each comprising a different base, and each comprising a coumarin-PEG-label of a different length. 
     
     
         43 . The method of  claim 14 , wherein the nucleic acid is incubated with four oligonucleotide-tagged ddNPPs, each comprising a different base, and each comprising an oligonucleotide of different length and/or composition. 
     
     
         44 . The method of  claim 43 , wherein the four coumarin-PEG labels are coumarin-PEG16, coumarin-PEG20, coumarin-PEG24, and coumarin-PEG36. 
     
     
         45 . The method of  claim 44 , wherein each coumarin-PEG label is attached to the terminal phosphate of the ddNPP. 
     
     
         46 . The method of  claim 44 , wherein each coumarin-PEG label is a coumarin-PEG-aminopropargyl label and is attached to the 5-position of the base if the ddNPP is ddCPP, ddUPP, or ddTPP, and to the 7-position of the base if the ddNPP is ddAPP or ddGPP. 
     
     
         47 . The method of any one of  claims 1 - 46 , wherein the sequence of the primer is 10-40 nucleotides long. 
     
     
         48 . The method of  claim 47 , wherein the sequence of the primer is 18-24 nucleotides long. 
     
     
         49 . An assay for performing the method of any one of  claims 1 - 48 . 
     
     
         50 . A dideoxynucleotide tetraphosphate (ddN4P) analogue, comprising a coumarin-polyethylene glycol (PEG)-aminopropargyl label attached to the terminal phosphate thereof. 
     
     
         51 . The ddN4P analogue of  claim 50 , having the structure 
       
         
           
           
               
               
           
         
         wherein n is 16, 20, 24, or 36, and wherein B is a base selected from the group consisting of adenine, cytosine, thymine, guanine, and uracil. 
       
     
     
         52 . The NPP analogue of  claim 50 , having the structure 
       
         
           
           
               
               
           
         
         wherein BASE is selected from the group adenine, cytosine, thymine, uracil, guanine, 7-deaza-adenine, and 7-deaza-guanine or analog thereof; 
         R and R′ can be independently H, OH, O-alkyl, F, Cl, Br, N 3 , NH 2 , O—NH 2 , O-allyl, O—CH 2 N 3 , 2′, 3′-isopropylidine or groups which only allow a single nucleotide to be incorporated by DNA polymerase; 
         TAG can be any polymeric molecule that can be detected by nanopore and may be selected from the group oligonucleotides, peptides, carbohydrates, and PEGs of different length. 
       
     
     
         53 . A composition comprising four ddN4P analogues of  claim 46  or  claim 51 , wherein each ddN4P comprises a different base and a distinct label, and each has a different value of n. 
     
     
         54 . The composition of  claim 53 , wherein the four ddN4P analogues are a ddA4P analogue, a ddG4P analogue, a ddC4P analogue, and either a ddT4P analogue or a ddU4P analogue. 
     
     
         55 . The ddN4P analogue of  claim 50 , wherein the base is guanine. 
     
     
         56 . A process for producing the ddN4P analogue of  claim 50 , comprising:
 a) contacting a ddN4P with diaminoheptane in carbodiimide (EDAC) and imidazole buffer under conditions permitting the diaminoheptane to attach to the terminal phosphate;   b) contacting a 6-methoxycoumarin N-hydroxysuccinimidyl ester (NHS) with an amino-PEG n -acid moiety in dimethylformamide, wherein n is the number of ethylene glycol monomers in the PEG, under conditions permitting the production of a coumarin-PEG n -acid compound;   c) reacting the product of step b) with N,N-disuccinimidyl carbonate in dimethylformamide, under conditions permitting the production of a coumarin-PEG n -NHS compound; and   d) reacting the products of steps a) and c) to produce a coumarin-PEG n -ddN4P analogue.   
     
     
         57 . The process of  claim 56 , further comprising, prior to step a), reacting a ddNTP with tributylammonium phosphate in order to obtain the ddN4P. 
     
     
         58 . A process for producing the ddG4P analogue of  claim 55 , comprising:
 a) contacting a ddG4P with diaminoheptane in carbodiimide (EDAC) and imidazole buffer under conditions permitting the diaminoheptane to attach to the terminal phosphate;   b) contacting a 6-methoxycoumarin N-hydroxysuccinimidyl ester (NHS) with an amino-PEG n -acid moiety in dimethylformamide, wherein n is the number of ethylene glycol monomers in the PEG, under conditions permitting the production of a coumarin-PEG n -acid compound;   c) reacting the product of step b) with N,N-disuccinimidyl carbonate in dimethylformamide, under conditions permitting the production of a coumarin-PEG n -NHS compound; and   d) reacting the products of steps a) and c) to produce a coumarin-PEG n -ddG4P analogue.   
     
     
         59 . The process of  claim 58 , further comprising, prior to step a), reacting a ddGTP with tributylammonium phosphate in order to obtain the ddG4P. 
     
     
         60 . A dideoxynucleotide triphosphate (ddNTP) analogue, comprising a coumarin-polyethylene glycol (PEG)-aminopropargyl label attached to the base thereof. 
     
     
         61 . The ddNTP analogue of  claim 60  having the structure 
       
         
           
           
               
               
           
         
         wherein B is a base selected from the group adenine, cytosine, thymine, guanine, and uracil; and n is 16, 20, 24, or 36. 
       
     
     
         62 . A composition comprising four ddNTP analogues of  claim 60  or  61 , wherein each ddNTP comprises a different base, and each has a different value of n. 
     
     
         63 . The composition of  claim 62 , wherein the four ddNTP analogues are a ddATP analogue, a ddGTP analogue, a ddCTP analogue, and either a ddTTP analogue or a ddUTP analogue. 
     
     
         64 . A process for producing a ddNTP analogue of  claim 60  or  61 , comprising:
 a) contacting a 6-methoxycoumarin N-hydroxysuccinimidyl ester (NHS) with an amino-PEG n -acid moiety in dimethylformamide, wherein n is the number of ethylene glycol monomers in the PEG, under conditions permitting the production of a coumarin-PEG-acid compound; 
 b) reacting the product of step a) with N,N-disuccinimidyl carbonate in dimethylformamide, under conditions permitting the production of a coumarin-PEG n -NHS compound; and 
 c) reacting the product of step b) with an aminopropargyl-ddNTP, wherein the aminopropargyl moiety is attached to the base, in dimethylformamide, to produce a coumarin-PEG n -aminopropargyl-ddNTP. 
 
     
     
         65 . The process of  claim 64 , wherein the aminopropargyl-ddNTP in step c) is a 5-aminopropargyl-ddNTP if the ddNTP comprises a cytosine, uracil, or thymidine base, and wherein the aminopropargyl-ddNTP in step c) is a 7-aminopropargyl-ddNTP if the ddNTP comprises a adenine or guanine base. 
     
     
         66 . The process of any one of  claims 56 - 59 ,  63 , and  65 , wherein n is 16, 20, 24, or 36. 
     
     
         67 . An alpha hemolysin protein, having a primer conjugated thereto. 
     
     
         68 . The alpha hemolysin protein of  claim 67 , wherein the alpha hemolysin comprises a C46 mutation, and the primer is conjugated to the cysteine residue at position 46. 
     
     
         69 . A process for producing the primer-conjugated α-hemolysin of  claim 68 , comprising:
 a) contacting a sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sSMCC) hetero bifunctional crosslinker, comprising an amino-reactive N-hydrosysuccinimide (NHS) ester and a thiol-reactive maleimide group at opposite ends, with a primer comprising a terminal amino group, under conditions permitting the terminal amino group to react with the amino-reactive NHS ester; 
 b) removing residual free sSMCCs from the solution; and 
 c) contacting the product resulting from step a) with an α-hemolysin, wherein the α-hemolysin comprises a C46 mutation, under conditions permitting the thiol-reactive maleimide group to react with the cysteine residue at position 46; 
 thereby conjugating the primer to the α-hemolysin. 
 
     
     
         70 . The process of  claim 69 , wherein the removal of residual free sSMCCs in step b) is performed via purification by gel filtration. 
     
     
         71 . A conductance measurement system comprising:
 a) an electrically resistive barrier separating at least a first and a second electrolyte solution;
 said electrically resistive barrier comprises at least one pore with a diameter on nanometer scale; 
 said at least one pore being configured to allow an ionic current to be driven across said first and second electrolyte solutions by an applied potential; 
   b) at least one labeled terminating nucleotide polyphosphate (NPP) analogue, wherein the label is attached to either the base or the terminal phosphate of the NPP analogue, in at least one of said first and second electrolyte solutions; and   c) a means of measuring the ionic current and a means of recording its time course as a time series, including time periods when the at least one pore is unobstructed by the label and also time periods when a label causes pulses of reduced-conductance.   
     
     
         72 . The system of  claim 71 , wherein said at least one pore comprises features configured to:
 (1) capture the polymer label on the primer-incorporated nucleotide that has the label attached to the base to produce a unique electronic signature, and   (2) detect the unique electronic signature of the polymer label while the labeled nucleotide is complexed with the polymerase and the primed template in a ternary complex before incorporation.   
     
     
         73 . The system of  claim 71  or  72 , wherein the labeled terminating NPP analogue is a ddNPP analogue selected from the group consisting of 
       
         
           
           
               
               
           
         
         wherein B is a base selected from the group adenine, cytosine, thymine, guanine, and uracil; and n is 16, 20, 24, or 36. 
       
     
     
         74 . The system of  claim 73 , comprising four ddNPP analogues having the structure 
       
         
           
           
               
               
           
         
         wherein each ddNTP comprises a different base, and each has a different value of n. 
       
     
     
         75 . The system of  claim 73 , comprising four ddNPP analogues having the structure 
       
         
           
           
               
               
           
         
         wherein each ddNTP comprises a different base, and each has a different value of n. 
       
     
     
         76 . The system of  claim 74  or  75 , wherein the four ddNTP analogues are a ddATP analogue, a ddGTP analogue, a ddCTP analogue, and either a ddTTP analogue or a ddUTP analogue.