Higher order structure and binding of peptide nucleic acids

Abstract

Peptide nucleic acids and analogues of peptide nucleic acids are used to form duplex, triplex, and other structures with nucleic acids and to modify nucleic acids. The peptide nucleic acids and analogues thereof also are used to modulate protein activity through, for example, transcription arrest, transcription initiation, and site specific cleavage of nucleic acids.

1 . A complex comprising a first nucleic acid strand and second and third strands; 
 said second and third strands independently containing a sequence of ligands covalently bound by linking moieties;    at least one of said second strand linking moieties comprising an amide, a thioamide, a sulfinamide or a sulfonamide linkage and a plurality of said ligands on said second strand interacting with said first strand; and    at least one of said third strand linking moieties comprising an amide, a thioamide, a sulfinamide or a sulfonamide linkage and a plurality of said ligands on said third strand interacting with said first strand or with said ligands on said second strand.    
     
     
         2 . The complex of  claim 1  wherein each of said second strand and said third strand, independently, include a plurality of monomeric units connected via amide, thioamide, sulfinamide or sulfonamide linkages.  
     
     
         3 . The complex of  claim 1  wherein said first strand is a DNA strand.  
     
     
         4 . The complex of  claim 1  wherein said first stand is a RNA strand.  
     
     
         5 . The complex of  claim 2  wherein said monomeric units are connected via amide linkages.  
     
     
         6 . The complex of  claim 2  wherein said monomeric units each include a first nitrogen that connects said ligands to said monomeric units and a second nitrogen atom that forms a portion of said amide, thioamide, sulfinamide or sulfonamide linkage.  
     
     
         7 . The complex of  claim 2  wherein a majority of said ligands of at least one of said second strand or third strand are pyrimidine bases.  
     
     
         8 . The complex of  claim 7  wherein said pyrimidine bases are selected from thymine and cytosine.  
     
     
         9 . The complex of  claim 2  wherein said nucleic acid strand is a purine rich strand.  
     
     
         10 . The complex of  claim 2  wherein said monomeric units comprise aminoethylglycine units.  
     
     
         11 . The complex of  claim 1  wherein said second and said third strands, independently, comprise a compound of the 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, heterocyclic moieties, and reporter ligands;  
 each of C 1 -C n  is (CR 6 R 7 ) 7  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 and R 7  are independently selected from the group consisting of hydrogen, (C 2 -C 5 )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 5 )alkyl, or R 5  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 5  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 greater than 2 but not more than 10;  
 each of G 1 -G n−1  is —NR 3 CO—, —NR 3 CS—, —NR 3 SO— or —NR 3 SO 2 —, in either orientation, where R 3  is as defined above;  
 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;  
                     
  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 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 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;  
 
 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, nucleosides, nucleotides, nucleotide diphosphates, nucleotide triphosphates, oligonucleotides, oligonucleosides and soluble and non-soluble polymers.  
 
     
     
         12 . The complex of  claim 1  wherein said second strand and said third strand, independently, comprise a compound of the formula III, IV or V:  
       
         
           
           
               
               
           
         
       
       wherein: 
 each L is independently selected from the group consisting of hydrogen, phenyl, heterocyclic moieties, 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 greater than 1,  
 each k, l, and m is, independently, zero or an integer from 1 to 5;  
 each p is zero or 1;  
 R h  is OH, NH 2  or —NHLysNH 2 ; and  
 R 1  is H or COCH 3 .  
 
     
     
         13 . The complex of  claim 1  wherein said interaction between said first and second strands occurs between heteocyclic bases of said first strand and ligands on said second strand.  
     
     
         14 . The complex of  claim 1  wherein said interaction between said first and third strands occurs between heteocyclic bases of said first strand and ligands on said third strand.  
     
     
         15 . The complex of  claim 1  wherein said interaction includes formation of non-covalent bonds.  
     
     
         16 . The complex of  claim 1  wherein said interaction of ligands is via hydrogen bonding.  
     
     
         17 . The complex of  claim 1  wherein said interaction between said first and second strands occurs between heteocyclic bases of said first strand and ligands on said second strand; 
 said interaction between said first and third strands occurs between heteocyclic bases of said first strand and ligands on said third strand; and    said interaction of ligands of said second strand and heterocyclic bases of said first strand is via Watson/Crick hydrogen bonding, and said interaction of ligands of said second strand with heterocyclic bases of said first strand is via one of Watson/Crick hydrogen bonding or Hoogsteen hydrogen bonding.    
     
     
         18 . A complex comprising a first nucleic acid strand, a second nucleic acid strand, and a third strand; 
 said third strand containing a sequence of ligands covalently bound by linking moieties and wherein a plurality of said ligands interact with at least one of said first strand and said second strand; and    at least one of said third strand linking moieties comprises an amide, a thioamide, a sulfinamide or asulfonamide linkage.    
     
     
         19 . The complex of  claim 18 , wherein said third strand includes a plurality of monomeric units that are connected via amide, thioamide, sulfinamide or sulfonamide backbone linkages.  
     
     
         20 . The complex of  claim 18  wherein said interaction includes formation of non-covalent bonds.  
     
     
         21 . The complex of  claim 20  wherein said interaction includes hydrogen bonding.  
     
     
         22 . The complex of  claim 18  wherein said interaction occurs between heteocyclic bases on said first strand or said second strand and ligands on said third strand.  
     
     
         23 . The complex of  claim 18  wherein said first strand and said second strand are DNA strands.  
     
     
         24 . The complex of  claim 23  wherein said DNA strands are double-stranded DNA.  
     
     
         25 . The complex of  claim 23  wherein said DNA strands are complementary DNA strands.  
     
     
         26 . The complex of  claim 19  wherein said monomeric units are connected via amide linkages.  
     
     
         27 . The complex of  claim 19  wherein said monomeric units each include a first nitrogen that connects said ligands to said monomeric units and a second nitrogen atom that forms a portion of said amide, thioamide, sulfinamide or sulfonamide linkage.  
     
     
         28 . The complex of  claim 18  wherein a majority of said ligands are pyrimidine bases.  
     
     
         29 . The complex of  claim 28  wherein said pyrimidine bases are selected from thymine and cytosine.  
     
     
         30 . The complex of  claim 18  wherein one of said nucleic acid strands is a purine rich strand.  
     
     
         31 . The complex of  claim 19  wherein said monomeric units comprise aminoethylglycine units.  
     
     
         32 . The complex of  claim 18  wherein said third strand comprises a compound of the 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, heterocyclic moieties, and reporter ligands;  
 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, aralkyl, heteroaryl, hydroxy, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkylthio, NR 3 R 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 5  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 greater than 2 but not more than 10;  
 each of G 1 -G n−1  is —NR 3 CO—, —NR 3 CS—, —NR 3 SO— or —NR 3 SO 2 —, in either orientation, where R 3  is as defined above;  
 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;  
                     
  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 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 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;  
 
 Q is —CO 2 H, —CONR′R″, —SO 2 H or —SO 2 NR′R″ or an activated derivative of —CO 2 H or —SO 2 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, nucleosides, nucleotides, nucleotide diphosphates, nucleotide triphosphates, oligonucleotides, oligonucleosides and soluble and non-soluble polymers.  
 
     
     
         33 . The complex of  claim 18  wherein said third strand comprises a compound of the formula III, IV or V:  
       
         
           
           
               
               
           
         
       
       wherein: 
 each L is independently selected from the group consisting of hydrogen, phenyl, heterocyclic moieties, 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 greater than 1,  
 each k, l, and m is, independently, zero or an integer from 1 to 5; 8  
 each p is zero or 1;  
 R h  is OH, NH 2  or —NHLysNH 2 ; and  
 R i  is H or COCH 3 .  
 
     
     
         34 . A process for modifying double-stranded DNA, comprising the steps of: 
 contacting said double-stranded DNA with a compound that comprises a sequence of ligands covalently bound by amide, thioamide, sulfinamide, or sulfonamide linking moieties, wherein a plurality of said ligands interact with said double-stranded DNA and thereby displace one of said DNA strands; and    modifying said displaced strand.    
     
     
         35 . The process of  claim 34  wherein modifying said displaced strand comprises cleaving said strand.  
     
     
         36 . The process of  claim 35  wherein said cleavage is effected by an enzyme.  
     
     
         37 . The process of  claim 36  wherein said double-stranded DNA is contacted with said compound intercellular and said enzymatic cleavage is effect by enzymes naturally present intercellular.  
     
     
         38 . The process of  claim 37  wherein said enzyme is a nuclease.  
     
     
         39 . The process of  claim 38  wherein said nuclease is nuclease S 1 .  
     
     
         40 . The process of  claim 34  wherein said compound further includes a moiety that modifies said displaced strand.  
     
     
         41 . The process of  claim 40  wherein moiety cleaves said displaced strand.  
     
     
         42 . The process of  claim 34  wherein a plurality of said ligands hydrogen bond with said DNA.  
     
     
         43 . The process of  claim 34  wherein said compound forms Watson-Crick hydrogen bonds with one strand of said double-stranded DNA.  
     
     
         44 . The process of  claim 31  wherein a first molecule of said compound forms Watson-Crick hydrogen bonds with a first strand of said double-stranded DNA and a second molecule of said compound forms Hoogsteen hydrogen bonds with said first strand.  
     
     
         45 . The process of  claim 34  wherein modifying said displaced strand comprises covalently bonding a bonding moiety to said displaced strand.  
     
     
         46 . The process of  claim 34  wherein modifying said displaced strand comprises activating cellular repair mechanisms to effects digestion of said displaced strand.  
     
     
         47 . The process of  claim 34  wherein said displaced strand is modified by covalently bonding a bonding moiety to said displaced stand, thereby initiating a cellular repair mechanism that effects digestion of said displaced strand.  
     
     
         48 . A process of inhibiting the expression of a gene comprising contacting said gene with a compound that comprises a sequence of ligands covalently bound by amide, thioamide, sulfinamide, or sulfonamide linking moieties, and wherein a plurality of said ligands interact with said gene.  
     
     
         49 . The process of  claim 48  wherein a plurality of said ligands hydrogen bond with DNA of said gene.  
     
     
         50 . The process of  claim 48  wherein inhibition of gene expression comprises transcription interference.  
     
     
         51 . The process of  claim 50  wherein said transcription interferences comprises RNA polymerase arrest.  
     
     
         52 . The process of  claim 51  wherein said transcription interference comprises transcription arrest outside of a promoter region of said gene.  
     
     
         53 . The process of  claim 49  wherein a plurality of said ligands hydrogen bond to a template strand of said gene.  
     
     
         54 . The process of  claim 48  wherein said compound includes at least six monomeric units connected via a polyamide backbone and each of said monomeric units includes one of said ligands.  
     
     
         55 . A process for arresting transcription of a gene comprising contacting said gene with a compound that comprises a sequence of ligands covalently bound by amide, thioamide, sulfinamide, or sulfonamide linking moieties, thereby binding a plurality of said ligands to a template DNA strand of said gene.  
     
     
         56 . A process for modulating the activity of a restriction enzyme at a DNA restriction site, comprising contacting said DNA with a compound that comprises a sequence of ligands covalently bound by amide, thioamide, sulfinamide, or sulfonamide linking moieties, thereby binding a plurality of said ligands to said DNA proximal to said restriction site.  
     
     
         57 . A process for sequencing DNA, comprising: 
 contacting said DNA with a compound that comprises a sequence of ligands covalently bound by amide, thioamide, sulfinamide, or sulfonamide linking moieties, thereby binding a plurality of said ligands to said DNA proximal to a DNA restriction site;    treating said DNA with a restriction enzyme that recognizes and cleaves said DNA at said restriction site; and    identifying at least one product of said cleavage.    
     
     
         58 . A process for inhibiting transcription of DNA comprising: 
 selecting DNA for which transcription is to be inhibited; and    adding to a mixture containing said DNA and a transcription factor for said DNA a compound that comprises a sequence of ligands covalently bound by aside, thioamide, sulfinamide, or sulfonamide linking moieties, thereby binding a plurality of said ligands to said-transcription factor.    
     
     
         59 . A method for modulating binding of RNA polymerase to double-stranded DNA comprising: 
 contacting said DNA with a compound that comprises a sequence of ligands covalently bound by amide, thioamide, sulfinamide, or sulfonamide linking moieties, thereby binding a plurality of said ligands to said DNA; and    exposing said complex to RNA polymerase.    
     
     
         60 . A process for initiating transcription of a gene comprising contacting said gene with a compound that comprises a sequence of ligands covalently bound by amide, thioamide, sulfinamide, or sulfonamide linking moieties, wherein a plurality of said ligands interact with said gene to melt double-stranded DNA of said gene and thereby expose a template strand of said DNA to recognition by RNA polymerase.  
     
     
         61 . The process of  claim 60  wherein said gene is contacted with said compound under conditions that form a hybrid strand with a non-coding strand of said DNA.  
     
     
         62 . The process of  claim 60  wherein said gene is contacted with said compound under conditions that form both a hybrid strand with said DNA and a template strand “D” loop.  
     
     
         63 . A process for initiating transcription of a gene, comprising contacting said gene with a compound that comprises a sequence of ligands covalently bound by amide, thioamide, sulfinamide, or sulfonamide linking moieties, wherein a plurality of said ligands interact with said gene to melt double-stranded DNA of said gene and thereby form a transcription elongation loop.  
     
     
         64 . A method for binding RNA polymerase to double-stranded DNA comprising: 
 contacting said DNA with a compound that comprises a sequence of ligands covalently bound by amide, thioamide, sulfinamide, or sulfonamide linking moieties, wherein a plurality of said ligands interact with said DNA; and    exposing said compound and said DNA to said RNA polymerase.    
     
     
         65 . A hybrid complex for modulating transcription comprising: 
 double-stranded DNA;    a compound that comprises a sequence of ligands covalently bound by amide, thioamide, sulfinamide, or sulfonamide linking moieties, wherein a plurality of said ligands interact with said double-stranded DNA; and    RNA polymerase in contact with at least one of said DNA and said compound.    
     
     
         66 . A synthetic transcription factor comprising: 
 double-stranded DNA; and    a compound that comprises a sequence of ligands covalently bound by amide, thioamide, sulfinamide, or sulfonamide linking moieties, wherein a plurality of said ligands interact with said double-stranded DNA.    
     
     
         67 . A sequence specific gene activator comprising a compound that comprises a sequence of ligands covalently bound by amide, thioamide, sulfinamide, or sulfonamide linking moieties, wherein a plurality of said ligands interact with a specific sequence of DNA of said gene.  
     
     
         68 . A sequence specific gene activator comprising first and second synthetic strands, wherein each strand comprises a sequence of about 6-50 ligands covalently bound by amide, thioamide, sulfinamide, or sulfonamide linking moieties, and each of said strands has a ligand sequence that recognizes a base sequence within proximal regions on at least one DNA strand of said gene such that said strands bind proximal to one another on said DNA strand.  
     
     
         69 . The gene activator of  claim 68  wherein the ligand sequence of said synthetic strands are complementary to a non-template strand of said DNA.  
     
     
         70 . The gene activator of  claim 68  wherein the ligand sequence of said synthetic strands is selected such that binding of said synthetic strands on said gene results in creation of a loop of DNA that includes the binding sites of said synthetic strands on said gene.  
     
     
         71 . A sequence specific gene activator comprising first and second synthetic strands, wherein: 
 each strand comprises a sequence of about 6-50 ligands covalently bound by amide, thioamide, sulfinamide, or sulfonamide linking moieties:    said first strand has a ligand sequence that recognizes a base sequence on one of the DNA strands of said gene;    said second strand has a ligand sequence that recognizes a base sequence on the other of the DNA strands of said gene; and    the recognized base sequence on said one DNA strand and the recognized base sequence of said other DNA strand are positioned proximal to one another on said gene.    
     
     
         72 . The gene activator of  claim 71  wherein ligand sequences of said synthetic strands is selected such that binding of said synthetic strands on said gene results in creation of a loop of DNA on said gene that includes the binding sites of said synthetic strands on said gene.  
     
     
         73 . A chimeric compound comprising a first strand section including DNA or RNA, and a second strand section including a sequence of ligands covalently bound by amide, thioamide, sulfinamide, or sulfonamide linking moieties, said first strand section and said second strand section being covalently bound.  
     
     
         74 . The chimeric structure of  claim 73  wherein said first stand section includes DNA.  
     
     
         75 . A double-stranded chimeric complex, wherein: 
 a first strand comprises a first strand section including DNA or RNA and a second strand section comprises a sequence of ligands covalently bound by amide, thioamide, sulfinamide, or sulfonamide linking moieties, said first and said second strand sections being covalently bound;    a second strand comprises DNA, RNA, or a sequence of ligands covalently bound by amide, thioamide, sulfinamide, or sulfonamide linking moieties; and    said first strand and said second strand interact with one another to form a duplex structure.    
     
     
         76 . A method for intercellularly modulating the activity of a transcription factor comprising: 
 forming a chimeric structure by covalently bonding a first strand section including DNA or RNA and a second strand section including a sequence of ligands covalently bound by amide, thioamide, sulfinamide, or sulfonamide linking moieties, wherein a plurality of said ligands bind with said transcription factor; and    introducing said chimeric structure into a cell.    
     
     
         77 . The method of  claim 76  further including selecting said ligands and said DNA or RNA such that a plurality of said ligands bind with the sugar-phosphate backbone of said RNA or DNA.  
     
     
         78 . A process for intercellularly inhibiting the binding of a protein comprising: 
 forming a compound that comprises a sequence of ligands covalently bound by amide, thioamide, sulfinamide, or sulfonamide linking moieties, wherein a plurality of said bind to said protein; and    introducing said structure into a cell.