Oligonucleotides containing pyrazolo[3,4-d]pyrimidines for hybridization and mismatch discrimination

Abstract

Oligonucleotides in which one or more purine residues are substituted by pyrazolo[3,4-d]pyrimidines exhibit improved hybridization properties. Oligonucleotides containing pyrazolo[3,4-d]pyrimidine base analogues have higher melting temperatures than unsubstituted oligonucleotides of identical sequence. Thus, in assays involving hybridization of an oligonucleotide probe to a target polynucleotide sequence, higher signals are obtained. In addition, mismatch discrimination is enhanced when pyrazolo[3,4-d]pyrimidine-containing oligonucleotides are used as hybridization probes, making them useful as probes and primers for hybridization, amplification and sequencing procedures, particularly those in which single- or multiple-nucleotide mismatch discrimination is required.

What is claimed is:  
     
         1 . A method for distinguishing polynucleotides with related sequences, the method comprising the following steps: 
 (a) providing an oligonucleotide having a defined sequence, wherein one or more purine residues of the oligonucleotide are substituted by a pyrazolo[3,4-d]pyrimidine;    (b) providing at least two polynucleotides, each of which comprises a target sequence, wherein one of the polynucleotides has a target sequence that is perfectly complementary to the oligonucleotide and at least one other of the polynucleotides has a related target sequence;    (c) separately incubating each of the polynucleotides with the oligonucleotide under hybridization conditions; and    (d) determining the degree of hybridization between the oligonucleotide and each of the polynucleotides.    
     
     
         2 . The method according to  claim 1 , wherein the oligonucleotide further cornprises a detectable label.  
     
     
         3 . The method according to  claim 2  wherein the detectable label is a fluorescent label.  
     
     
         4 . The method according to  claim 3  wherein the label is a fluorescein.  
     
     
         5 . The method according to  claim 3  wherein the oligonucleotide comprises multiple fluorescent labels.  
     
     
         6 . The method according to  claim 5  wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels.  
     
     
         7 . The method according to  claim 3  wherein the oligonucleotide further comprises a quenching agent which quenches the fluorescence emission of the fluorescent label.  
     
     
         8 . The method according to  claim 7  wherein the fluorescent label is a fluorescein.  
     
     
         9 . The method according to  claim 8  wherein the quenching agent is tetramethylrhodamine.  
     
     
         10 . The method according to  claim 7 , further comprising the step of altering the spatial relationship between the fluorescent label and the quenching agent subsequent to hybridization.  
     
     
         11 . The method according to  claim 10 , wherein alteration of the spatial relationship between the fluorescent label and the quenching agent is accomplished by exonuclease hydrolysis of the oligonucleotide.  
     
     
         12 . The method according to  claim 1 , wherein release of label occurs as a result of exonuclease hydrolysis.  
     
     
         13 . The method according to  claim 12 , wherein the degree of hybridization between the oligonucleotide and each of the polynucleotides is determined by the quantity of label that is released from the oligonucleotide subsequent to hybridization.  
     
     
         14 . The method according to  claim 1 , wherein the polynucleotides differ by a single nucleotide.  
     
     
         15 . The method according to  claim 12 , wherein the polynucleotides differ by a single nucleotide.  
     
     
         16 . The method according to  claim 1 , wherein one or more guanine residues are substituted by 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one.  
     
     
         17 . The method according to  claim 1 , wherein one or more adenine residues are substituted by 4-amino-1H-pyrazolo[3,4-d]pyrimidine.  
     
     
         18 . The method according to  claim 1 , wherein one or more purine residues are substituted by 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one.  
     
     
         19 . The method according to  claim 1 , wherein one or more purine residues are substituted by hypoxanthine.  
     
     
         20 . The method according to  claim 1 , wherein the oligonucleotide further comprises one or more minor groove binder (MGB) moieties.  
     
     
         21 . The method according to  claim 20 , wherein the minor groove binder moiety is selected from the group consisting of the trimer of 3-carbamoyl-1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI 3 ) and the pentamer of N-methylpyrrole-4-carbox-2-amide (MPC 5 ).  
     
     
         22 . The method according to  claim 1 , wherein the oligonucleotide is less than 21 nucleotides in length.  
     
     
         23 . The method according to  claim 1 , wherein the degree of hybridization between the oligonucleotide and each of the polynucleotides is determined by the priming ability of the oligonucleotide.  
     
     
         24 . The method according to  claim 23 , wherein priming occurs as part of an amplification reaction.  
     
     
         25 . The method according to  claim 24 , wherein the amplification reaction is a polymerase chain reaction.  
     
     
         26 . The method according to  claim 3 , wherein more than one oligonucleotide is used.  
     
     
         27 . The method according to  claim 26 , wherein two oligonucleotides are used.  
     
     
         28 . The method according to  claim 27 , wherein the first of the two oligonucleotides comprises a fluorescence donor and the second of the two oligonucleotides comprises a fluorescence acceptor, and further wherein the emission wavelengths of the fluorescence donor overlap the absorption wavelengths of the fluorescence acceptor.  
     
     
         29 . A method for detecting the presence of a target sequence in a polynucleotide, the method comprising the following steps: 
 (a) providing a polynucleotide which is to be tested for the presence of the target sequence;    (b) providing an oligonucleotide having a sequence that is substantially complementary to the target sequence;    (c) incubating the polynucleotide and the oligonucleotide under hybridization conditions; and    (d) identifying hybridized nucleic acids;    wherein one or more purine residues of the oligonucleotide are substituted by a pyrazolo[3,4-d]pyrimidine.    
     
     
         30 . The method according to  claim 29 , wherein multiple polynucleotides are tested for the presence of the target sequence, and wherein the polynucleotides have related target sequences.  
     
     
         31 . The method according to  claim 30 , wherein the polynucleotides differ from one another by a single nucleotide within the target sequence.  
     
     
         32 . The method according to  claim 31 , wherein the oligonucleotide further comprises a minor groove binding moiety.  
     
     
         33 . The method according to  claim 32 , wherein the minor groove binding moiety is selected from the group consisting of the trimer of 3-carbamoyl-1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI 3 ) and the pentarner of N-methylpyrrole-4-carbox-2-amide (MPC 5 ).  
     
     
         34 . The method according to  claim 29  wherein the oligonucleotide is a primer comprising an extendible 3′-hydroxyl group.  
     
     
         35 . The method according to  claim 34 , wherein hybridized nucleic acids are identified by extending the primer with a polymerizing enzyme.  
     
     
         36 . The method according to  claim 35  wherein the polymerizing enzyme is a thermostable enzyme.  
     
     
         37 . The method according to  claim 34 , wherein the oligonucleotide is a primer in an amplification reaction.  
     
     
         38 . The method according to  claim 37 , wherein the amplification reaction is a polymerase chain reaction.  
     
     
         39 . A method for primer extension, the method comprising the following steps: 
 (a) providing a polynucleotide containing a target sequence,    (b) providing one or more oligonucleotide primers complementary to the target sequence,    (c) providing a polymerizing enzyme and nucleotide substrates, and    (d) incubating the polynucleotide, the oligonucleotide primers, the enzyme and the substrates under conditions favorable for polymerization;    wherein one or more purine residues of the one or more oligonucleotide primers are substituted by a pyrazolo[3,4-d]pyrimidine.    
     
     
         40 . The method according to  claim 39 , wherein the method is part of an amplification reaction.  
     
     
         41 . The method according to  claim 40 , wherein the amplification reaction is a polymerase chain reaction.  
     
     
         42 . The method according to  claim 39  wherein the method is used in the synthesis of a cDNA molecule.  
     
     
         43 . A method for determining the nucleotide sequence of a polynucleotide, the method comprising the following steps: 
 (a) providing an array of oligonucleotides having different known sequences,    (b) incubating the polynucleotide with the array under hybridization conditions, and    (c) determining to which of the oligonucleotides in the array the polynucleotide hybridizes;    wherein one or more purine residues in each of the oligonucleotides are substituted by a pyrazolo[3,4-d]pyrimidine.    
     
     
         44 . A method for determining the nucleotide sequence of a target sequence in a polynucleotide, the method comprising the following steps: 
 (a) providing a polynucleotide comprising the target sequence;    (b) providing at least two oligonucleotides of known sequence wherein one or more purine residues of the oligonucleotides are substituted by a pyrazolo[3,4-d]pyrimidine, and wherein one of the at least two oligonucleotides has a sequence that is perfectly complementary to the target sequence and at least one other of the oligonucleotides has a related target sequence;    (c) separately incubating each of the oligonucleotides with the polynucleotide under hybridization conditions; and    (d) determining the degree of hybridization between each of the oligonucleotides and the polynucleotide.    
     
     
         45 . The method according to  claim 44 , wherein the at least one other oligonucleotide has a single-nucleotide mismatch with the target sequence  
     
     
         46 . A method for examining gene expression in a cell, the method comprising the following steps: 
 (a) providing a population of polynucleotides representative of the genes expressed in the cell,    (b) providing an array of oligonucleotides of different sequences,    (c) incubating the population of polynucleotides with the array under hybridization conditions, and    (d) determining which of the oligonucleotides in the array become hybridized to polynucleotides;    wherein one or more purine residues in each of the oligonucleotides are substituted by a pyrazolo[3,4-d]pyrimidine.    
     
     
         47 . A method for identifying a mutation in a target sequence of a gene of interest, the method comprising the following steps: 
 (a) providing a polynucleotide that comprises the target sequence,    (b) providing an array of oligonucleotides of different sequences, wherein the different sequences include the wild-type target sequence and different mutant target sequences,    (c) incubating the polynucleotide with the array under hybridization conditions, and    (d) determining which of the oligonucleotides in the array become hybridized to the polynucleotide;    wherein one or more purine residues in each of the oligonucleotides are substituted by a pyrazolo[3,4-d]pyrimidine.