Modulation of the binding properties of nucleic acid binding partners

Abstract

A method for specifically determining the presence of a target by contacting the target in the presence of other targets with a probe capable of binding to the target to form a complex of the target with the probe, where the contacting is performed in the presence of a compound enhancing the specificity of the binding or the complex formed is contacted with a compound enhancing the specificity of the binding. This method opens the way to a better discrimination between related nucleic acid sequences in diagnostic assays.

1 . A method for specifically determining the presence of at least one single stranded target nucleic acid in the presence of at least one other nucleic acid sequence, comprising contacting a single stranded target nucleic acid with a probe in the presence of a compound which enhances the specificity of binding, wherein said probe binds to said target nucleic acid to form a complex between said target nucleic acid and probe, or contacting a target nucleic acid with a probe which binds to said target nucleic acid to form a complex between said target nucleic acid and probe and contacting said complex with a compound which enhances the specificity of binding, wherein said target nucleic acid, said probe or both said target nucleic acid and said probe are peptide nucleic acid binding partners and wherein said target nucleic acid and probe or said complex is in a solution when contacted with said compound. 
     
     
         2 . The method according to  claim 1 , wherein said compound is soluble in aqueous solutions. 
     
     
         3 . The method according to  claim 1 , wherein said compound has a hydrophobic part or both a hydrophobic part and a hydrophilic part. 
     
     
         4 . The method according to  claim 1 , wherein said compound is a surfactant or detergent. 
     
     
         5 . The method according to  claim 4 , wherein said compound is sodium dodecyl sulphate. 
     
     
         6 . The method according to  claim 1 , wherein said compound is positively charged. 
     
     
         7 . The method according to  claim 1 , wherein said target nucleic acid and probe or said complex is contacted with said compound in a solution containing salts at a concentration between 5 to 50 mM. 
     
     
         8 . The method according to  claim 1 , wherein the compound is present in a concentration of between 0.05 and 0.5% w/v. 
     
     
         9 . A method for enhancing the specificity of binding of a single stranded target nucleic acid to a peptide nucleic acid binding partner, comprising adding a compound which enhances the specificity of binding to a reaction mixture containing the single stranded target nucleic acid and the peptide nucleic acid binding partner, wherein said target nucleic acid differs from other nucleic acids in said reaction mixture by at least one position. 
     
     
         10 . The method according to  claim 9 , wherein said compound is selected from the group consisting of surfactants and detergents. 
     
     
         11 . A method for lowering the assay temperature for the specific binding of a nucleic acid to a peptide nucleic acid binding partner in a reaction mixture, comprising adding a compound selected from the group consisting of surfactants and detergents to the reaction mixture in an amount effective to lower the assay temperature for the specific binding of a single stranded target nucleic acid to the peptide nucleic acid binding partner. 
     
     
         12 . A method for increasing the velocity of binding of a nucleic acid to a peptide nucleic acid binding partner, comprising adding a compound selected from the group consisting of surfactants and detergents to a reaction mixture containing nucleic acid and the binding partner in an amount effective to increase the velocity of binding of a single stranded target nucleic acid to the peptide nucleic acid binding partner. 
     
     
         13 . The method according to  claim 9 , wherein the peptide nucleic acid binding partner binds more strongly than to an analogous nucleic acid of the same length and sequence to a target nucleic acid. 
     
     
         14 . The method according to  claim 1 , wherein said peptide nucleic acid binding partners have the general formula 1: 
       
         
           
           
               
               
           
         
         wherein 
         n is at least 2, 
         each of L 1 -L n  is independently selected from the group consisting of hydrogen, hydroxy, (C 1 -C 4 ) alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups, heterocyclic moieties, reporter ligands and chelating moieties; 
         each of C 1 -C n  is (CR 6 R 7 )y where R 6  is hydrogen and R 7  is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R 6  and R 7  are independently selected from the group consisting of hydrogen, (C 2 -C 6 )alkyl, aryl, (C 1 -C 4 )aralkyl, heteroaryl, hydroxy, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkylthio, NR 3 R 4  and SR 5 , where R 3  and R 4  are as defined below, and R 5  is hydrogen, (C 1 -C 6 )alkyl, hydroxy, (C 1 -C 4 )alkoxy, or (C 1 -C 4 )alkylthio-substituted (C 1  to C 6 )alkyl or R 6  and R 7  taken together complete an alicyclic or heterocyclic system; 
         each of D 1 -D n  is (CR 6 R 7 ) z  where R 6  and R 7  are as defined above; 
         each of y and z is zero or an integer from 1 to 10, the sum y+z being at least 2, but not more than 10; 
         each of G 1 -G n−1  is NR 3 CO—, —NR 3 C 5 —, NR 3 SO— or NR 3 SO 2 —, where R 3  is as defined below; 
         each of A 1 -A n  and B 1 -B n  are selected such that: 
         (a) A is a group of formula (IIa), (IIb), (IIc) or (IId), and B is N or R 3 N+; or 
         (b) A is a group of formula (IId) and B is CH; 
       
       
         
           
           
               
               
           
         
         wherein; 
         X is O, S, Se, NR 3 , CH 2  or C(CH 3 ) 2 ; 
         Y is a single bond, O, S or NR 4 ; 
         each of p and q is zero or an integer from 1 to 5, the sum p+q being not more than 10; 
         each of r and s is zero or an integer from 1 to 5, the sum r+s being not more than 10; 
         each R 1  and R 2  is independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl which may be hydroxy- or (C 1 -C 4 )alkoxy- or (C 1 -C 4 )alkylthio-substituted, hydroxy, (C 1 -C 4 )alkoxy, (C 1 -C 4 )alkylthio, amino and halogen; and 
         each R 3  and R 4  is independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl, hydroxy- or (C 1 -C 4 )alkoxy- or (C 1 -C 4 )alkylthio-substituted, (C 1 -C 4 )alkyl, hydroxy, (C 1 -C 4 )alkoxy, (C 1 -C 4 )alkylthio and amino; and 
         Q is CO 2 H, —CONR′R″, —SO 3 H or —SO 2 —NR′R″ or an activated derivative of —CO 2 H or —SO 3 H, and 
         I is —NR′R″ wherein R′ and R″ are independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, nucleosides, nucleotides, nucleotide diphosphates, nucleotide triphosphates, oligonucleotides, including both oligoribonucleotides and oligodeoxyribonucleotides, oligonucleosides and soluble and non-soluble polymers. 
       
     
     
         15 . The method according to  claim 9 , wherein said peptide nucleic acid binding partners have the general formula 1: 
       
         
           
           
               
               
           
         
         wherein 
         n is at least 2, 
         each of L 1 -L n  is independently selected from the group consisting of hydrogen, hydroxy, (C 1 -C 4 )alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups, heterocyclic moieties, reporter ligands and chelating moieties; 
         each of C 1 —C n  is (CR 6 R 7 ) y  where R 6  is hydrogen and R 7  is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R 6  and R 7  are independently selected from the group consisting of hydrogen, (C 2 -C 6 )alkyl, aryl, (C 1 -C 4 )aralkyl, heteroaryl, hydroxy, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkylthio, NR 3 R 4  and SR 5 , where R 3  and R 4  are as defined below, and R 5  is hydrogen, (C 1 -C 6 )alkyl, hydroxy, (C 1 -C 4 )alkoxy, or (C 1 -C 4 )alkylthio-substituted (C 1  to C 6 )alkyl or R 6  and R 7  taken together complete an alicyclic or heterocyclic system; 
         each of D 1 -D n  is (CR 6 R 7 ) z  where R 6  and R 7  are as defined above; 
         each of y and z is zero or an integer from 1 to 10, the sum y+z being at least 2, but not more than 10; 
         each of G 1 -G n−1  is NR 3 CO—, —NR 3 C 5 —, NR 3 SO— or NR 3 SO 2 —, where R 3  is as defined below; 
         each of A 1 -A n  and B 1 -B n  are selected such that: 
         (a) A is a group of formula (IIa), (IIb), (IIc) or (IId), and B is N or R 3 N+; or 
         (b) A is a group of formula (IId) and B is CH; 
       
       
         
           
           
               
               
           
         
         wherein; 
         X is O, S, Se, NR 3 , CH 2  or C(CH 3 ) 2 ; 
         Y is a single bond, O, S or NR 4 ; 
         each of p and q is zero or an integer from 1 to 5, the sum p+q being not more than 10; 
         each of r and s is zero or an integer from 1 to 5, the sum r+s being not more than 10; 
         each R 1  and R 2  is independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl which may be hydroxy- or (C 1 -C 4 )alkoxy- or (C 1 -C 4 )alkylthio-substituted, hydroxy, (C 1 -C 4 )alkoxy, (C 1 -C 4 )alkylthio, amino and halogen; and 
         each R 3  and R 4  is independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl, hydroxy- or (C 1 -C 4 )alkoxy- or (C 1 -C 4 )alkylthio-substituted, (C 1 -C 4 )alkyl, hydroxy, (C 1 -C 4 )alkoxy, (C 1 -C 4 )alkylthio and amino; and 
         Q is CO 2 H, —CONR′R″, —SO 3 H or —SO 2 —NR′R″ or an activated derivative of —CO 2 H or —SO 3 H, and 
         I is —NR′R″ wherein R′ and R″ are independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, nucleosides, nucleotides, nucleotide diphosphates, nucleotide triphosphates, oligonucleotides, including both oligoribonucleotides and oligodeoxyribonucleotides, oligonucleosides and soluble and non-soluble polymers. 
       
     
     
         16 . The method according to  claim 1 , wherein both said target nucleic acid and said probe are peptide nucleic acid binding partners. 
     
     
         17 . The method according to  claim 9 , wherein both said stranded target nucleic acid and said peptide nucleic acid binding partner are peptide nucleic acid binding partners. 
     
     
         18 . The method according to  claim 11 , wherein both said nucleic acid and said peptide nucleic acid are peptide nucleic acid binding partners. 
     
     
         19 . The method according to  claim 12 , wherein both said nucleic acid and said peptide nucleic acid binding partner are peptide nucleic acid binding partners.