Use of nucleic acid analogues in diagnostics and analytical procedures

Abstract

The present invention pertains to certain nucleic acid analogs and related kits that are useful for the capture, recognition, detection, identification, or quantification of certain chemical or biological entities.

1 - 26 . (canceled)  
     
     
         27 . A nucleic acid analogue for use in the capture, recognition, detection, identification, or quantification of one or more chemical or microbiological entities, which analogue is selected from the group consisting of: 
 (a) a peptide nucleic acid (PNA) comprising a polyamide backbone bearing a plurality of ligands at respective spaced locations along said backbone, said ligands being each independently naturally occurring nucleobases, non-naturally occurring nucleobases, or nucleobase-binding groups, each said ligand being bound directly or indirectly to a nitrogen atom in said backbone, and said ligand bearing nitrogen atoms mainly being separated from one another in said backbone by from 4 to 8 intervening atoms;    (b) a nucleic acid analogue containing a peptide nucleic acid (PNA) capable of hybridizing to a nucleic acid of complementary sequence to form a hybrid that is more stable against denaturation by heat than a hybrid between the conventional deoxyribonucleotide corresponding to said analogue and said nucleic acid; and    (c) a nucleic acid analogue containing a peptide nucleic acid (PNA) capable of hybridizing to a double stranded nucleic acid in which one strand has a sequence complementary to said analogue, so as to displace the other strand from said one strand;    wherein said nucleic acid analogue is either immobilized with a solid support or has incorporated within said nucleic acid analogue, or conjugated to said nucleic acid analogue, a detectable label.    
     
     
         28 . A nucleic acid analogue as claimed in  claim 27 , having the general formula:  
       
         
           
           
               
               
           
         
       
       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, and reporter ligands, at least one of L 1 -L n  being a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, or a nucleobase-binding group;  
 each of A 1 -A n  is a single bond, a methylene group, or a group of formula:  
                     
 where:  
 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 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 alkoxy- or alkylthio-substituted, hydroxy, alkoxy, 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 alkoxy- or alkylthio-substituted (C 1 -C 4 )alkyl, hydroxy, alkoxy, alkylthio, and amino;  
 each of B 1 —B n  is N or R 3 N + , where R 3  is defined above;  
 each of C 1 —C n  is CR 6 R 7 , CHR 6 CHR 7 , or CR 6 R 7 CH 2 , 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 )alkylthio, NR 3 R 4 , and SR 5 , where R 3  and R 4  are as defined above, and R 5  is hydrogen, (C 1 -C 6 )alkyl, hydroxy-, alkoxy-, or alkylthio-substituted (C 1 -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 , CH 2 CR 6 R 7 , or CHR 6 CHR 7 , where R 6 R 7  are as defined above;  
 each of G 1 -G n  is —CONR 3 —, —CSNR 3 —, —SONR 3 —, or —SO 2 NR 3 —, in either orientation, where R 3  is as defined above;  
 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 —NHR″′R″″ or —NR″′C(O)R″″, where R′, R″, R″′, and R″″ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers.  
 
     
     
         29 . A nucleic acid analogue as claimed in  claim 28 , having the general formula:  
       
         
           
           
               
               
           
         
       
       wherein, 
 each L is independently selected from the group consisting of hydrogen, phenyl, naturally occurring nucleobases, and non-naturally occurring nucleobases;  
 each R 7′  is independently selected from the group consisting of hydrogen and the side chains of naturally occurring alpha amino acids;  
 n is an integer from 1 to 60;  
 each k and m is, independently, zero or one;  
 each 1 is independently from zero to five;  
 R h  is —OH, —NH 2 , or —NHLysNH 2 ; and  
 R i  is —H or —COCH 3 .  
 
     
     
         30 . A nucleic acid analogue as claimed in  claim 29 , having the general formula:  
       
         
           
           
               
               
           
         
       
       wherein, 
 each L is independently selected from the group consisting of the nucleobases thymine, adenine, cytosine, guanine, and uracil;  
 each R 7′  is hydrogen; and  
 n is an integer from 1 to 30.  
 
     
     
         31 . A nucleic acid analogue as claimed in  claim 27 , incorporating or conjugated to a detectable label.  
     
     
         32 . The nucleic acid analogue of  claim 31 , wherein said label is a radio isotope label, a biotin, a fluorophore, a chemiluminescence label, an antigen, an antibody, or a spin label.  
     
     
         33 . A nucleic acid analogue as claimed in  claim 27 , immobilised to a solid support.  
     
     
         34 . The nucleic acid analogue of  claim 33 , incorporated in an affinity capture column.  
     
     
         35 . A peptide nucleic acid (PNA) for use in the capture, recognition, detection, identification, or quantification of one or more chemical or microbiological entities, wherein said PNA comprises a polyamide backbone bearing a plurality of ligands at respective spaced locations along said backbone, said ligands being each independently naturally occurring nucleobases, non-naturally occurring nucleobases, or nucleobase-binding groups, each said ligand being bound directly or indirectly to a nitrogen atom in said backbone, and said ligand bearing nitrogen atoms mainly being separated from one another in said backbone by from 4 to 8 intervening atoms, and 
 wherein said PNA is either immobilized with a solid support or has incorporated within said PNA, or conjugated to said PNA, a detectable label.    
     
     
         36 . A PNA as claimed in  claim 35 , having the general formula:  
       
         
           
           
               
               
           
         
       
       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 and reporter ligands, at least one of L 1 -L n  being a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, or a nucleobase-binding group; each of A 1 -A n  is a single bond, a methylene group, or a group of formula:  
                     
 where:  
 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 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 alkoxy- or alkylthio-substituted, hydroxy, alkoxy, 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 alkoxy- or alkylthio-substituted (C 1 -C 4 )alkyl, hydroxy, alkoxy, alkylthio, and amino;  
 each of B 1 —B n  is N or R 3 N + , where R 3  is defined above;  
 each of C 1 —C n  is CR 6 R 7 , CHR 6 CHR 7 , or CR 6 R 7 CH 2 , 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 )alkylthio, NR 3 R 4 , and SR 5 , where R 3  and R 4  are as defined above, and R 5  is hydrogen, (C 1 -C 6 )alkyl, hydroxy-, alkoxy-, or alkylthio-substituted (C 1 -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 , CH 2 CR 6 R 7 , or CHR 6 CHR 7 , where R 6  R 7  are as defined above; each of G 1 -G n  is —CONR 3 —, —CSNR 3 —, —SONR 3 —, or —SO 2 NR 3 —, in either orientation, where R 3  is as defined above;  
 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 —NHR″′R″″ or —NR″′C(O)R″″, where R′, R″, R″′, and R″″ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers.  
 
     
     
         37 . A PNA as claimed in  claim 36 , having the general formula:  
       
         
           
           
               
               
           
         
       
       wherein, 
 each L is independently selected from the group consisting of hydrogen, phenyl, naturally occurring nucleobases, and non-naturally occurring nucleobases;  
 each R 7′  is independently selected from the group consisting of hydrogen and the side chains of naturally occurring alpha amino acids;  
 n is an integer from 1 to 60;  
 each k and m is, independently, zero or one;  
 each 1 is independently from zero to five;  
 R h  is —OH, —NH 2 , or —NHLysNH 2 ; and  
 R i  is —H or —COCH 3 .  
 
     
     
         38 . A PNA as claimed in  claim 37 , having the general formula:  
       
         
           
           
               
               
           
         
       
       wherein, 
 each L is independently selected from the group consisting of the nucleobases thymine, adenine, cytosine, guanine, and uracil;  
 each R 7′  is hydrogen; and  
 n is an integer from 1 to 30.  
 
     
     
         39 . A nucleic acid analogue containing a peptide nucleic acid (PNA) for use in the capture, recognition, detection, identification, or quantification of one or more chemical or microbiological entities, 
 wherein said nucleic acid analogue is capable of hybridizing to a nucleic acid of complementary sequence to form a hybrid that is more stable against denaturation by heat than a hybrid between the conventional deoxyribonucleotide corresponding to said analogue and said nucleic acid, and wherein said nucleic acid analogue is either immobilized with a solid support or has incorporated within said nucleic acid analogue, or conjugated to said nucleic acid analogue, a detectable label.    
     
     
         40 . A nucleic acid analogue as claimed in  claim 39 , having the general formula:  
       
         
           
           
               
               
           
         
       
       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 and reporter ligands, at least one of L 1 -L n  being a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, or a nucleobase-binding group;  
 each of A 1 -A n  is a single bond, a methylene group, or a group of formula:  
                     
 where:  
 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 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 alkoxy- or alkylthio-substituted, hydroxy, alkoxy, 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 alkoxy- or alkylthio-substituted (C 1 -C 4 )alkyl, hydroxy, alkoxy, alkylthio, and amino;  
 each of B 1 -B n  is N or R 3 N + , where R 3  is defined above;  
 each of C 1 -C n  is CR 6 R 7 , CHR 6 CHR 7 , or CR 6 R 7 CH 2 , 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 )alkylthio, NR 3 R 4 , and SR 5 , where R 3  and R 4  are as defined above, and R 5  is hydrogen, (C 1 -C 6 )alkyl, hydroxy-, alkoxy-, or alkylthio-substituted (C 1 -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 , CH 2 CR 6 R 7 , or CHR 6 CHR 7 , where R 6  R 7  are as defined above;  
 each of G 1 -G n  is —CONR 3 —, —CSNR 3 —, —SONR 3 —, or —SO 2 NR 3 —, in either orientation, where R 3  is as defined above;  
 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 —NHR″′R″″ or —NR″′C(O)R″″, where R′, R″, R″′, and R″″ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers.  
 
     
     
         41 . A nucleic acid analogue as claimed in  claim 40 , having the general formula:  
       
         
           
           
               
               
           
         
       
       wherein, 
 each L is independently selected from the group consisting of hydrogen, phenyl, naturally occurring nucleobases, and non-naturally occurring nucleobases;  
 each R 7′  is independently selected from the group consisting of hydrogen and the side chains of naturally occurring alpha amino acids;  
 n is an integer from 1 to 60;  
 each k and m is, independently, zero or one;  
 each 1 is independently from zero to five;  
 R h  is —OH, —NH 2 , or —NHLysNH 2 ; and  
 R i  is —H or —COCH 3 .  
 
     
     
         42 . A nucleic acid analogue as claimed in  claim 41 , having the general formula:  
       
         
           
           
               
               
           
         
       
       wherein, 
 each L is independently selected from the group consisting of the nucleobases thymine, adenine, cytosine, guanine, and uracil;  
 each R 7′  is hydrogen; and  
 n is an integer from 1 to 30.  
 
     
     
         43 . A nucleic acid analogue containing a peptide nucleic acid (PNA) for use in the capture, recognition, detection, identification, or quantification of one or more chemical or microbiological entities, 
 wherein said analogue is capable of hybridizing to a double stranded nucleic acid in which one strand has a sequence complementary to said analogue, so as to displace the other strand from said one strand, and    wherein said nucleic acid analogue is either immobilized with a solid support or has incorporated within said nucleic acid analogue, or conjugated to said nucleic acid analogue, a detectable label.    
     
     
         44 . A nucleic acid analogue as claimed in  claim 43 , having the general formula:  
       
         
           
           
               
               
           
         
       
       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 and reporter ligands, at least one of L 1 -L n  being a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, or a nucleobase-binding group;  
 each of A 1 -A n  is a single bond, a methylene group, or a group of formula:  
                     
 where:  
 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 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 alkoxy- or alkylthio-substituted, hydroxy, alkoxy, 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 alkoxy- or alkylthio-substituted (C 1 -C 4 )alkyl, hydroxy, alkoxy, alkylthio, and amino;  
 each of B 1 —B n  is N or R 3 N + , where R 3  is defined above;  
 each of C 1 —C n  is CR 6 R 7 , CHR 6 CHR 7 , or CR 6 R 7 CH 2 , 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 )alkylthio, NR 3 R 4 , and SR 5 , where R 3  and R 4  are as defined above, and R 5  is hydrogen, (C 1 -C 6 )alkyl, hydroxy-, alkoxy-, or alkylthio-substituted (C 1 -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 , CH 2 CR 6 R 7 , or CHR 6 CHR 7 , where R 6  R 7  are as defined above;  
 each of G 1 -G n  is —CONR 3 —, —CSNR 3 —, —SONR 3 —, or —SO 2 NR 3 —, in either orientation, where R 3  is as defined above;  
 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 —NHR″′R″″ or —NR″′C(O)R″″, where R′, R″, R″′, and R″″ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers.  
 
     
     
         45 . A nucleic acid analogue as claimed in  claim 44 , having the general formula:  
       
         
           
           
               
               
           
         
       
       wherein, 
 each L is independently selected from the group consisting of hydrogen, phenyl, naturally occurring nucleobases, and non-naturally occurring nucleobases;  
 each R 7′  is independently selected from the group consisting of hydrogen and the side chains of naturally occurring alpha amino acids;  
 n is an integer from 1 to 60;  
 each k and m is, independently, zero or one;  
 each 1 is independently from zero to five;  
 R h  is —OH, —NH 2 , or —NHLysNH 2 ; and  
 R i  is —H or —COCH 3 .  
 
     
     
         46 . A nucleic acid analogue as claimed in  claim 45 , having the general formula:  
       
         
           
           
               
               
           
         
       
       wherein, 
 each L is independently selected from the group consisting of the nucleobases thymine, adenine, cytosine, guanine, and uracil;  
 each R 7′  is hydrogen; and  
 n is an integer from 1 to 30.