Nucleic Acid displacer compositions and cells comprising same

Abstract

Procedures and compositions for forming and stabilizing non stable branch migrated oligo- and polydeoxynucleotides utilizing a displacer which is either single stranded or partially double stranded and hybridized to a linker strand. The displacer may contain one or more modified nucleotides.

1 . In a method of forming a non stable complex between one strand of a recipient polydeoxynucleotide sequence and a displacer sequence of single stranded DNA where the displacer sequence is at least partially complementary to such strand of a recipient polydeoxynucleotide sequence, the improvement comprising stabilizing the complex.  
     
     
         2 . The method of  claim 1  wherein the nonstable complex is stabilized by the presence of at least one modified nucleotide in the displacer strand.  
     
     
         3 . The method of  claim 1  wherein the complex is stabilized by forming a DNA triplex between the displacer sequence and the recipient duplex.  
     
     
         4 . The method of  claim 1  wherein the displacer strand comprises a nucleotide sequence and a sequence specific DNA binding moiety that does not significantly melt the recipient DNA duplex at the site to which it attaches.  
     
     
         5 . The method of  claim 1  wherein the displacer sequence is duplexed with a linker in a displacer-linker duplex wherein the displacer-linker duplex comprises two strands; 
 1. a displacer strand of which a portion comprises nucleotides complementary to one strand of a recipient polydeoxynucleotide duplex and a portion which comprises a sequence complementary to and hybridized with a linker strand, and    2. a linker strand complementary to and hybridized with the displacer strand.    
     
     
         6 . An oligo- or polydeoxynucleotide displacer-linker duplex which is capable of initiating branch migration at the end of a recipient polydeoxynucleotide duplex without the prior formation of a stable hybrid with such recipient polydeoxynucleotide duplex, which displacer-linker duplex comprises two strands: 
 a. a displacer strand of which a portion comprises nucleotides complementary to one strand of a recipient polydeoxynucleotide duplex and a portion which comprises a sequence complementary to and hybridized with a linker strand, and    b. a linker strand complementary to and hybridized with the displacer strand.    
     
     
         7 . The displacer-linker duplex of  claim 6  which can initiate branch migration at a restriction endonuclease cleavage site.  
     
     
         8 . The displacer-linker duplex of  claim 6  which can hybridize to and initiate branch migration adjacent to a 3′ or 5′ single stranded extension on the recipient polydeoxynucleotide duplex.  
     
     
         9 . The displacer-linker duplex of  claim 6  wherein at least one of the nucleotides complementary to one strand of the recipient polydeoxynucleotide duplex is modified to increase the stability of the hybrid displacer-recipient duplex.  
     
     
         10 . The displacer-linker duplex of  claim 6  wherein at least one of the nucleotides complementary to one strand of a recipient polydeoxynucleotide duplex is modified to increase the melting temperature of the hybrid displacer-recipient duplex.  
     
     
         11 . The displacer-linker duplex of  claim 9  wherein the modified nucleotide is selected from the group consisting of modified nucleotides which increase the association constant with the complementary deoxynucleotide by at least about 20 percent.  
     
     
         12 . The displacer-linker duplex of  claim 9  wherein the modified nucleotide is selected from the group consisting of 5-halogenated pyrimidine nucleotides, 5-methyldeoxycytidine, diaminopurine deoxynucleotide, ribonucleotides, and 2′-alkylated ribonucleotides.  
     
     
         13 . The displacer-linker duplex of  claim 9  wherein the modified nucleotide is a 5-halogenated pyrimidine nucleotide.  
     
     
         14 . The displacer-linker duplex of  claim 9  wherein the modified nucleotide is 5-bromodeoxyuridine.  
     
     
         15 . The displacer-linker duplex of  claim 9  wherein the modified nucleotide is 5-methyldeoxycytidine.  
     
     
         16 . The displacer-linker duplex of  claim 6  which contains a modification which permits detection of the displacer-recipient hybrid.  
     
     
         17 . The modified displacer-linker duplex of  claim 16  wherein the modification is selected from the group consisting of radioactive labels, fluorescent labels and targets for detection, including biotin moieties, enzymes and phosphorothioate linkages.  
     
     
         18 . The modified displacer-linker duplex of  claim 16  wherein the modification is present in the linker.  
     
     
         19 . The displacer-linker duplex of  claim 6  which contains a modification which allows capture of the displacer-recipient hybrid by affinity chromatography.  
     
     
         20 . The displacer-linker duplex of  claim 19  wherein the modification is selected from the group consisting of biotin moieties and phosphorothioate linkages.  
     
     
         21 . The displacer-linker duplex of  claim 19  wherein the modification is present in the linker.  
     
     
         22 . The displacer-linker duplex of  claim 6  which also comprises a 5′ or 3′ single-stranded extension complementary to a 5′ or 3′ single-stranded extension resulting from the digestion of a polydeoxynucleotide duplex with a restriction endonuclease.  
     
     
         23 . An artificially constructed polydeoxynucleotide hybrid comprising a naturally occurring recipient polydeoxynucleotide duplex hybridized to the displacer-linker duplex of any of claims  6 - 22 .  
     
     
         24 . The artificially constructed polydeoxynucleotide hybrid of any of claims  6 - 22  wherein the linker strand is covalently linked to one of the strands of the recipient duplex.  
     
     
         25 . An artificially constructed polydeoxynucleotide hybrid comprising a naturally occurring recipient polydeoxynucleotide duplex hybridized to the displacer of claims  57 - 87 .  
     
     
         26 . A method of modifying a recipient polydeoxynucleotide duplex by contacting such recipient polydeoxynucleotide duplex with a displacer-linker duplex under conditions that permit the formation of a hybrid polydeoxynucleotide duplex, wherein 
 the displacer-linker duplex comprises two strands; 
 1. a displacer strand of which a portion comprises nucleotides complementary to one strand of a recipient polydeoxynucleotide duplex and a portion which comprises a sequence complementary to and hybridized with a linker strand, and  
 2. a linker strand complementary to and hybridized with the displacer strand.  
   
     
     
         27 . The method of  claim 26  wherein the recipient duplex terminates in a 3′ or 5′ single stranded extension and the displacer strand contains a sequence complementary to the extension.  
     
     
         28 . The method of  claim 26  where the hybrid is stabilized after formation of the hybrid polynucleotide duplex.  
     
     
         29 . The method of  claim 28  wherein the hybrid is stabilized by covalently linking the linker strand of the displacer-linker duplex to the strand of the recipient duplex complementary to the displacer strand.  
     
     
         30 . The method of  claim 29  wherein the covalent link is a phosphodiester linkage.  
     
     
         31 . The method of  claim 29  wherein the hybrid is stabilized by ligating the linker strand of the displacer-linker duplex to the strand of the recipient duplex complementary to the displacer strand using T4 DNA ligase.  
     
     
         32 . The method of  claim 26  wherein at least one of the nucleotides complementary to one strand of the recipient polydeoxynucleotide duplex is a modified nucleotide which increases the stability of the hybrid displacer-recipient duplex.  
     
     
         33 . The method of  claim 26  wherein at least one of the nucleotides complementary to one strand of the recipient polydeoxynucleotide duplex is a modified nucleotide which increases the melting temperature of the hybrid displacer-recipient duplex.  
     
     
         34 . The method of  claim 32  wherein the modified nucleotide is selected from the group consisting of modified nucleotides which increase the association constant with the complementary deoxynucleotide by at least about 20 percent.  
     
     
         35 . The method of  claim 32  wherein the modified nucleotide is selected from the group consisting of 5-halogenated pyrimidine nucleotides, 5-methyldeoxycytidine, diaminopurine deoxynucleotide, ribonucleotides, and 2′-alkylated ribonucleotides.  
     
     
         36 . The method of  claim 32  wherein the modified nucleotide is a 5-halogenated pyrimidine nucleotide.  
     
     
         37 . The method of  claim 32  wherein the modified nucleotide is 5-bromodeoxycytidine.  
     
     
         38 . The method of  claim 32  wherein the modified nucleotide is 5-methyldeoxycytidine.  
     
     
         39 . The method of  claim 32  wherein from about 10 percent to about 80 percent of the nucleotides complementary to one strand of the recipient polydeoxynucleotide duplex are modified nucleotides.  
     
     
         40 . A method of labelling an artificially constructed nucleic acid hybrid of a naturally occurring recipient polydeoxynucleotide duplex hybridized to a displacer-linker duplex which is capable of initiating branch migration at the end of the recipient polydeoxynucleotide duplex without the prior formation of a stable hybrid with such recipient polydeoxynucleotide duplex, which displacer-linker duplex comprises two strands; 
 1. a displacer strand of which a portion comprises nucleotides complementary to one strand of a recipient polydeoxynucleotide duplex and a portion which comprises a sequence complementary to and hybridized with a linker strand, and    2. a linker strand complementary to and hybridized with the displacer strand,    which method of labelling comprises modifying the displacer-linker duplex to incorporate therein a modification which permits detection of the artificially constructed nucleic acid hybrid.    
     
     
         41 . The method of  claim 40  wherein the displacer-linker duplex is modified prior to hybridization with the naturally occurring recipient polydeoxynucleotide duplex.  
     
     
         42 . The method of  claim 40  wherein the linker strand of the displacer-linker duplex is covalently linked to the strand of the naturally occurring recipient polydeoxynucleotide duplex complementary to the displacer strand.  
     
     
         43 . The method of  claim 40  wherein the modification is selected from the group consisting of radioactive labels, fluorescent labels, enzymes and chemical labels including biotin moieties and phosphorothioate linkages.  
     
     
         44 . The method of  claim 40  wherein the modification is selected from the group consisting of targets for affinity chromatography.  
     
     
         45 . The method of  claim 44  wherein the modification is selected from the group consisting of biotin moieties and phosphorothioate linkages.  
     
     
         46 . The method of  claim 40  wherein the modification comprises a 5′ or 3′ single-stranded extension of the displacer-linker duplex, which extension 
 1) is unaffected by formation of the displacer-linker-recipient polydeoxynucleotide duplex hybrid, and    2) is a target for attachment to polydeoxynucleotide duplexes containing complementary 5′ or 3′ single stranded extensions.    
     
     
         47 . The method of  claim 40  used to label one end of a cloned deoxynucleotide insert in a vector.  
     
     
         48 . The method of  claim 47  where the vector is a plasmid vector.  
     
     
         49 . The method of  claim 47  where the vector is a cosmid vector.  
     
     
         50 . The method of  claim 47  where the vector is a yeast artificial chromosome vector.  
     
     
         51 . In a method of restriction endonuclease mapping of an insert, the improvement comprising labelling the insert by the method of  claim 40 .  
     
     
         52 . In a method of capture of an artificially constructed nucleic acid hybrid by affinity chromatography, the improvement comprising labelling the hybrid by the method of  claim 40 .  
     
     
         53 . In a method of enriching a recipient polydeoxynucleotide duplex in a population of polydeoxynucleotide duplexes, the improvement comprising labelling the recipient polydeoxynucleotide duplex by the method of  claim 40 .  
     
     
         54 . In a method of covalently attaching a restriction endonuclease linker onto a recipient polydeoxynucleotide duplex, the improvement comprising labelling the resulting hybrid by the method of  claim 40 .  
     
     
         55 . In a method of selectively cloning a recipient polydeoxynucleotide duplex by covalently attaching a restriction endonuclease linker onto such recipient polydeoxynucleotide duplex, the improvement comprising labelling the hybrid by the method of  claim 40 .  
     
     
         56 . In a method of isolating clones of contiguous polydeoxyribonucleotide duplexes by covalently attaching a restriction endonuclease linker onto the duplexes, the improvement comprising labelling the clones by the method of  claim 40 .  
     
     
         57 . An oligo- or polydeoxynucleotide displacer which is capable of binding to a recipient polydeoxynucleotide duplex which displacer comprises 
 1) a first sequence which is capable of initiating triple helix formation, and which comprises 
 a) at least six consecutive pyrimidine bases or  
 b) at least seven bases where at least six of the bases are pyrimidine bases and the seventh base is guanine, and  
   2) a second sequence proximate to such first sequence which is complementary to and runs antiparallel to the second strand of the recipient duplex and which is capable of initiating branch migration proximate to the triple helix.    
     
     
         58 . The displacer of  claim 57  wherein the second sequence is adjacent to the first sequence.  
     
     
         59 . The displacer of  claim 57  wherein the second sequence is separated from the first sequence by from 1 to 5 intervening moieties.  
     
     
         60 . The displacer of  claim 59  wherein the intervening moieties are nucleotides.  
     
     
         61 . The displacer of  claim 60  wherein at least one of the moieties is a modified nucleotide.  
     
     
         62 . The displacer of  claim 59  wherein wherein one of the intervening moieties has an intercalating agent covalently attached.  
     
     
         63 . The displacer of  claim 57  wherein at least one of the nucleotides complementary to one strand of the recipient polydeoxynucleotide duplex is modified to increase the stability of the displacer-recipient complex.  
     
     
         64 . The displacer of  claim 57  wherein at least one of the nucleotides complementary to one strand of a recipient polydeoxynucleotide duplex is modified to increase the melting temperature of the displacer-recipient complex.  
     
     
         65 . The displacer of  claim 63  wherein the modified nucleotide is selected from the group consisting of modified nucleotides which increase the association constant with the complementary deoxynucleotide by at least about  20  percent.  
     
     
         66 . The displacer of  claim 63  wherein the modification is in the first sequence.  
     
     
         67 . The displacer of  claim 66  wherein the modified nucleotide is a 5-halogenated pyrimidine nucleotide.  
     
     
         68 . The displacer of  claim 66  wherein the modified nucleotide is selected from the group consisting of 5-bromodeoxyuridine and 5-methyldeoxycytidine.  
     
     
         69 . The displacer of  claim 63  wherein the modification is in the second sequence.  
     
     
         70 . The displacer of  claim 69  wherein the modified nucleotide is selected from the group consisting of 5-halogenated pyrimidine nucleotides, 5-methyldeoxycytidine, diaminopurine deoxynucleotide, ribonucleotides and a 2′-alkylated ribonucleotides.  
     
     
         71 . The displacer of  claim 69  wherein the modified nucleotide is a 5-halogenated pyrimidine nucleotide.  
     
     
         72 . The displacer of  claim 69  wherein the modified nucleotide is 5-bromodeoxyuridine.  
     
     
         73 . The displacer of  claim 69  wherein the modified nucleotide is 5-methyldeoxycytidine.  
     
     
         74 . The displacer of  claim 57  which further comprises at least one moiety attached to a terminus of the oligo or polydeoxynucleotide, which moiety confers endonuclease resistance to the terminus to which it is attached.  
     
     
         75 . The displacer of  claim 74  wherein the moiety is attached to the deoxyribose moiety of a terminal nucleotide.  
     
     
         76 . The displacer of  claim 75  wherein the moiety is indirectly attached to the deoxyribose moiety of a terminal nucleotide.  
     
     
         77 . The displacer of  claim 74  wherein the moiety is attached to the hydroxyl group of a terminal nucleotide.  
     
     
         78 . The displacer of  claim 74  wherein the moiety is attached to the phosphate moiety of a terminal nucleotide.  
     
     
         79 . The displacer of  claim 74  where the moiety is selected from the group consisting of intercalating agents, isoureas, carbodiimides and N-hydroxybenzotriazoles.  
     
     
         80 . The displacer of  claim 77  wherein the moiety is a methylthiophosphonate.  
     
     
         81 . The displacer of  claim 74  wherein the moiety is a selected from the group consisting of polypeptides and proteins.  
     
     
         82 . The displacer of  claim 74  wherein the moiety is a 2′,3′-dideoxyribose nucleotide attached to the 3′-terminal deoxyribonucleotide through a phosphodiester linkage.  
     
     
         83 . The displacer of  claim 82  wherein the 2′,3′-dideoxyribose nucleotide is a modified 2′,3′-dideoxyribose nucleotide.  
     
     
         84 . The displacer of  claim 57  which contains a modification which permits detection of the displacer-recipient hybrid.  
     
     
         85 . The modified displacer of  claim 84  wherein the modification is selected from the group consisting of radioactive labels, fluorescent labels, enzymes and targets for detection, including biotin moieties and phosphorothioate linkages.  
     
     
         86 . The displacer of  claim 57  which contains a modification which allows capture of the displacer-recipient hybrid by affinity chromatography.  
     
     
         87 . The displacer of  claim 86  wherein the modification is selected from the group consisting of biotin moieties and phosphorothioate linkages.  
     
     
         88 . An method of modifying a recipient polydeoxynucleotide duplex comprising contacting such recipient polydeoxynucleotide duplex with the displacer of  claim 57  under conditions that permit the formation of a complex.  
     
     
         89 . The method of  claim 88  wherein at least one of the nucleotides complementary to one strand of the recipient polydeoxynucleotide duplex is modified to increase the stability of the displacer-recipient complex.  
     
     
         90 . The method of  claim 88  wherein at least one of the nucleotides complementary to one strand of a recipient polydeoxynucleotide duplex is modified to increase the melting temperature of the displacer-recipient complex.  
     
     
         91 . The method of  claim 88  wherein the modified nucleotide is selected from the group consisting of modified nucleotides which increase the association constant with the complementary deoxynucleotide by at least about 20 percent.  
     
     
         92 . The method of  claim 88  wherein the modification is in the first sequence of the displacer of  claim 57 .  
     
     
         93 . The method of  claim 92  wherein the modified nucleotide is selected from the group consisting of 5-bromodeoxyuridine and 5-methyldeoxycytidine.  
     
     
         94 . The method of  claim 88  wherein the modification is in the second sequence of the displacer of  claim 57 .  
     
     
         95 . The method of  claim 94  wherein the modified bases are selected from the group consisting of 5-halogenated pyrimidine nucleotides, 5-methyldeoxycytidine, diaminopurine deoxynucleotide, ribonucleotides, and 2′-alkylated ribonucleotides.  
     
     
         96 . The method of  claim 94  wherein the modified nucleotide is a 5-halogenated pyrimidine nucleotide.  
     
     
         97 . The method of  claim 94  wherein the modified nucleotide is 5-bromodeoxyuridine.  
     
     
         98 . The method of  claim 94  wherein the modified nucleotide is 5-methyldeoxycytidine.  
     
     
         99 . The method of  claim 88  wherein the displacer contains at least one moiety attached to a terminus of the oligo or polynucleotide, which moiety confers endonuclease resistance to the terminus to which it is attached.  
     
     
         100 . The method of  claim 99  wherein the moiety is attached to the deoxyribose moiety of a terminal nucleotide.  
     
     
         101 . The method of  claim 100  wherein the moiety is indirectly attached to the deoxyribose moiety of a terminal nucleotide.  
     
     
         102 . The method of  claim 99  wherein the moiety is attached to the hydroxyl group of a terminal nucleotide.  
     
     
         103 . The method of  claim 99  wherein the moiety is attached to the phosphate moiety of a terminal nucleotide.  
     
     
         104 . The method of  claim 99  where the moiety is selected from the group consisting of intercalating agents, isoureas, carbodiimides and N-hydroxybenzotriazoles.  
     
     
         105 . The method of  claim 101  wherein the moiety is a methylthiophosphonate.  
     
     
         106  The method of  claim 99  wherein the moiety is a selected from the group consisting of polypeptides and proteins.  
     
     
         107 . The method of  claim 99  wherein the moiety is a 2′,3′-dideoxyribose nucleotide attached to the 3′-terminal deoxyribonucleotide through a phosphodiester linkage.  
     
     
         108 . The method of  claim 107  wherein the 2′,3′-dideoxyribose nucleotide is a modified 2′,3′-dideoxyribose nucleotide.  
     
     
         109 . A method of labelling a displacer-recipient complex comprising contacting a recipient polydeoxynucleotide duplex with the displacer of  claim 101  under conditions that permit the formation of a complex wherein the displacer contains a modification which will permit detection of the displacer-recipient complex.  
     
     
         110 . The method of  claim 109  wherein the modification is selected from the group consisting of radioactive labels, fluorescent and chemiluminescent labels, enzymes and targets for detection.  
     
     
         111 . The method of  claim 109  wherein the modification is selected from the group consisting of targets for affinity chromatography.  
     
     
         112 . The method of  claim 111  wherein the modification is selected from the group consisting of biotin moieties and phosphorothioate linkages.  
     
     
         113 . In a method of capture of an artificially constructed nucleic acid hybrid by affinity chromatography, the improvement comprising modifying the hybrid by the method of  claim 88 .  
     
     
         114 . In a method of enriching a recipient polydeoxynucleotide duplex in a population of polydeoxynucleotide duplexes, the improvement comprising labelling the recipient polydeoxynucleotide duplex by the method of  claim 88 .  
     
     
         115 . In a method for the site specific addition, deletion or alteration of nucleotides in a recipient polydeoxynucleotide duplex, the improvement comprising modifying the duplex by the method of  claim 88 .  
     
     
         116 . In a method of repairing a mutational lesion comprising replacing a naturally occurring strand of DNA with a modified strand of DNA, the improvement wherein the new strand is introduced to the naturally occurring duplex by the method of  claim 88  and displaces the original strand.