Pna diagnostic use

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 . A nucleic acid analogue for use in the capture, recognition, detection, identification or quantitation of one or more chemical or microbiological entities, which analogue is
 (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 capable of hybridising to a nucleic acid of complementary sequence to form a hybrid which is more stable against denaturation by heat than a hybrid between the conventional deoxyribonucleotide corresponding to said analogue and said nucleic acid; or   (c) a nucleic acid analogue capable of hybridising 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.   
     
     
         2 . A nucleic acid analogue as claimed in  claim 1 , 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  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 as 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 )alkyl, aryl, 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 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  and R 7  are as defined above; 
 each of G 1 -G n-1  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. 
 
     
     
         3 . A nucleic acid analogue as claimed in  claim 2 , 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; and each 1 is independently from zero to 5; 
 R h  is OH, NH 2  or —NHLysNH 2 ; and 
 R i  is H or COCH 3 . 
 
     
     
         4 . A nucleic acid analogue as claimed in  claim 3 , 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. 
 
     
     
         5 . A nucleic acid analogue as claimed in any one of  claims 1  to  4 , incorporating or conjugated to a detectable label. 
     
     
         6 . A labelled nucleic acid analogue as claimed in  claim 5 , wherein said label is a radio isotope label, an enzyme label, biotin, a fluorophore, a chemiluminescence label, an antigen, an antibody or a spin label. 
     
     
         7 . The use of nucleic acid analogue as defined in any one of  claims 1  to  6  in the capture, recognition, detection, identification or quantitation of one or more chemical or microbiological entities. 
     
     
         8 . A method of capturing a nucleic acid comprising/contacting under hybridising conditions said nucleic acid with a nucleic acid analogue as claimed in any one of  claims 1  to  6  immobilised to a solid support, which nucleic acid analogue has a sequence of ligands suitable to hybridise to said nucleic acid. 
     
     
         9 . A method as claimed in  claim 8 , wherein said captured nucleic acid is detected, recognised, quantitated or identified by treatment with a nucleic acid recognition agent whilst bound to said immobilised nucleic acid analogue. 
     
     
         10 . A method as claimed in  claim 9 , wherein the captured nucleic acid has a first region hybridised to said immobilised nucleic acid analogue and a second region which is not so hybridised and is treated with a labelled nucleic acid or nucleic acid analogue which is adapted to hybridise to at least part of said second region and said label is detected. 
     
     
         11 . A method as claimed in  claim 8 , for capturing a mRNA wherein said immobilised nucleic acid analogue comprises sequential ligands hybridisable to poly A tails of said mRNA to capture said mRNA. 
     
     
         12 . A method as claimed in  claim 11 , wherein said sequential ligands are thymine. 
     
     
         13 . A method as claimed in any one of  claims 8  or  11  or  12 , wherein said nucleic acid once captured is released from said immobilised nucleic acid analogue by subjecting the immobilised nucleic acid analogue and captured nucleic acid to dehybridising conditions. 
     
     
         14 . A nucleic acid analogue as claimed in any one of  claims 1  to  6 ; immobilised to a solid support. 
     
     
         15 . An immobilised nucleic acid analogue as claimed in  claim 14 , incorporated in an affinity capture column. 
     
     
         16 . A method of recognition, detection or quantitation of a target nucleic acid comprising hybridising said target to a labelled nucleic acid analogue as claimed in  claim 5  or  claim 6  of sufficiently complementary sequence to hybridise therewith under hybridising conditions and detecting or quantitating said label of the nucleic acid analogue so hybridised to said target. 
     
     
         17 . A method as claimed in  claim 16 , wherein said target nucleic acid is immobilised on a substrate prior to said hybridisation. 
     
     
         18 . A method as claimed in  claim 17 , wherein said target nucleic acid is immobilised to said substrate by the hybridisation of a first region thereof to a capture nucleic acid or nucleic acid analogue having a sequence sufficiently complementary to said first region to hybridise therewith and which is itself immobilised to said substrate and wherein said labelled nucleic acid analogue hybridises to a second region of said target. 
     
     
         19 . A method for displacing one strand from a nucleic acid duplex comprising hybridising to said duplex a nucleic acid analogue having an affinity for the other strand of said duplex sufficient to be able to displace said one strand therefrom. 
     
     
         20 . A method of detecting, identifying or quantitating a double stranded target nucleic acid comprising hybridising thereto a displacing nucleic acid analogue capable of displacing one strand from a double stranded target in which the other strand is of complementary sequence to said displacing nucleic acid analogue, wherein said displacing nucleic acid analogue is of sufficiently complementary sequence to said other strand of said double stranded target to hybridise thereto so as to displace said one strand of said target in single stranded form, and detecting or quantitating the presence of said one displaced strand. 
     
     
         21 . A method as claimed in  claim 20 , wherein the displaced strand is broken down into fragments and the presence of said fragments is detected. 
     
     
         22 . A method as claimed in  claim 21 , wherein said displaced strand is broken down by attack by a nuclease. 
     
     
         23 . A kit for use in a diagnostics procedure and comprising at least one labelled nucleic acid analogue as claimed in  claim 5  or  claim 6 , and at least one detection reagent for detecting said labelled nucleic acid analogue. 
     
     
         24 . A kit as claimed in  claim 23 , further including a nucleic acid analogue as claimed in any one of  claims 1  to  4  immobilised on a solid support. 
     
     
         25 . A kit comprising an immobilised nucleic acid analogue as claimed in any one of  claims 1  to  4 , in combination with at least one nucleic acid recognition agent for detecting the presence of nucleic acid captured in use by said nucleic acid analogue. 
     
     
         26 . A kit as claimed in  claim 25 , wherein said nucleic acid recognition agent is a labelled nucleic acid or a labelled nucleic acid analogue.