Sequence specific detection of nucleic acids using a solid carrier bound with nucleic acid analog probes

A method for the sequence-specific determination of mutations or polymorphisms in a nucleic acid, said method comprising:

• contacting a sample that contains the nucleic acid to be determined with a basically planar solid carrier comprising at least two single stranded peptide nucleic acid probes of different base sequence covalently bound to said solid carrier, and wherein at least one peptide nucleic acid probe binds to nucleic acid in said sample under conditions where the nucleic acid to be determined in said sample binds to the peptide nucleic acid probe, and wherein at least one of said peptide nucleic acid probes has a base sequence that is complementary to the base sequence of the nucleic acid to be determined, and wherein at least one additional peptide nucleic acid probe has a base sequence that is not complementary to the base sequence of the nucleic acid to be determined, and
• determining in which region the nucleic acid to be determined binds to the peptide nucleic acid probes that are bound to said carrier, as an indication of the mutation or polymorphism of the nucleic acid to be determined;
• wherein the probes are nucleic acid analogs that have the general formula (I):
• wherein:
• n is at least 2,
• each of L1-Ln is independently selected from the group consisting of hydrogen, hydroxy, (C1-C4) alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups and reporter ligands, at least one of L1-Ln being a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, or a nucleobase-binding group;
• each of A1 through An is a single bond, a methylene group or a group of formula (IIa) or (IIb):
• wherein in (IIa) or (IIb):
• X is O, S, Se, NR3, CH2 or C(CH3)2;
• Y is a single bond, O, S or NR4;
• 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 ss is zero or an integer from 1 to 5, the sum r+ss being not more than 10;
• each R1 and R2 is independently selected from the group consisting of hydrogen, (C1-C4) alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen;
• and
• each R3 and R4 is independently selected from the group consisting of hydrogen, (C1-C4) alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C1-C4)alkyl, hydroxy, alkoxy, alkylthio and amino;
• and wherein in formula (I);
• each of B1-Bn is N or R3N+, where R3 is as defined above;
• each of C1-Cn is CR6R7,CHR6CHR7or CR6R7CH2, wherein R6 is hydrogen and R7 is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R6 and R7 are independently selected from the group consisting of hydrogen, (C2-C6) alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C1-C6) alkoxy, (C1-C6) alkylthio, NR3R4 and SR5, where R3 and R4 are as defined above, and R5 is hydrogen, (C1-C6) alkyl, hydroxy-, alkoxy-, or alkylthio- substituted (C1-C6) alkyl, or R6 and R7 taken together complete an alicyclic or heterocyclic system;
• each of D1-Dn is CR6R7, CH2CR6R7 or CHR6CHR7, where R6 and R7 are as defined above;
• each of G1-Gn-1 is
• in either orientation, wherein R3 is as defined above;
• Q is —CO2H, —CONR′R″, —SO3H or SO2NR′R″ or an activated derivative of —CO2H, —SO3H;
• 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, wherein C and D can optionally be CHR6(CH2)sssCHR7 where sss is from 0 to 2.

A method for the selective detection of nucleic acid mutants, said method comprising:

• contacting a sample containing mutant nucleic acid, in the presence of a large amount of non-mutant nucleic acid, with a basically planar solid carrier comprising at least two single stranded peptide nucleic acid probes of different base sequence, each probe being covalently bound to a separate and distinct region of the solid carrier, and wherein at least one peptide nucleic acid probe binds to nucleic acid in said sample under conditions where nucleic acid binds to the peptide nucleic acid probe;
• and
• determining in the regions where the nucleic acid mutants, the non-mutant nucleic acids or both the nucleic acid mutants and the non-mutant nucleic acids bind to said solid carrier wherein the probes are nucleic acid analogs that have the general formula (I):
• wherein:
• n is at least 2,
• each of L1-Ln is independently selected from the group consisting of hydrogen, hydroxy, (C1-C4) alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups and reporter ligands, at least one of L1-Ln being a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, or a nucleobase-binding group;
• each of A1 through An is a single bond, a methylene group or a group of formula (IIa) or (IIb):
• wherein in (IIa) or (IIb):
• X is O, S, Se, NR3, CH2 or C(CH3)2;
• Y is a single bond, O, S or NR4;
• 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 ss is zero or an integer from 1 to 5, the sum r+ss being not more than 10;
• each R1 and R2 is independently selected from the group consisting of hydrogen, (C1-C4) alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen;
• and
• each R3 and R4 is independently selected from the group consisting of hydrogen, (C1-C4) alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C1-C4)alkyl, hydroxy, alkoxy, alkylthio and amino;
• and wherein in formula (I);
• each of B1-Bn is N or R3N+, where R3 is as defined above;
• each of C1-Cn is CR6R7, CHR6CHR7or CR6R7CH2, wherein R6 is hydrogen and R7 is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R6 and R7 are independently selected from the group consisting of hydrogen, (C2-C6) alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C1-C6) alkoxy, (C1-C6) alkylthio, NR3R4 and SR5, where R3 and R4 are as defined above, and R5 is hydrogen, (C1-C6) alkyl, hydroxy-, alkoxy-, or alkylthio- substituted (C1-C6) alkyl, or R6 and R7 taken together complete an alicyclic or heterocyclic system;
• each of D1-Dn is CR6R7, CH2CR6R7 or CHR6CHR7, where R6 and R7 are as defined above;
• each of G1-Gn-1 is
• in either orientation, wherein R3 is as defined above;
• Q is —CO2H, —CONR′R″, —SO3H or SO2NR′R″ or an activated derivative of —CO2H, —SO3H;
• 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, wherein C and D can optionally be CHR6(CH2)sssCHR7 where sss is from 0 to 2.

A method for determining the relative amounts of a mutant nucleic acid and a non-mutant nucleic acid in a sample, said method comprising:

• contacting a nucleic acid-containing sample with a basically planar solid carrier comprising at least two single stranded peptide nucleic acid probes of different base sequence, each probe being covalently bound to a separate and distinct region of the solid carrier, and wherein at least two peptide nucleic acid probes each bind to different nucleic acids in said sample under conditions where nucleic acid binds to the peptide nucleic acid probe;
• and
• determining the relative amount of mutant and non-mutant nucleic acid in said sample by comparing the amount of the mutant nucleic acid with the amount of the non-mutant nucleic acid, each being bound at different regions of the solid carrier wherein the probes are nucleic acid analogs that have the general formula (I):
• wherein:
• n is at least 2,
• each of L1-Ln is independently selected from the group consisting of hydrogen, hydroxy, (C1-C4) alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups and reporter ligands, at least one of L1-Ln being a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, or a nucleobase-binding group;
• each of A1 through An is a single bond, a methylene group or a group of formula (IIa) or (IIb):
• wherein in (IIa) or (IIb):
• X is O, S, Se, NR3, CH2 or C(CH3)2;
• Y is a single bond, O, S or NR4;
• 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 ss is zero or an integer from 1 to 5, the sum r+ss being not more than 10;
• each R1 and R2 is independently selected from the group consisting of hydrogen, (C1-C4) alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen;
• and
• each R3 and R4 is independently selected from the group consisting of hydrogen, (C1-C4) alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C1-C4)alkyl, hydroxy, alkoxy, alkylthio and amino;
• and wherein in formula (I);
• each of B1-Bn is N or R3N+, where R3 is as defined above;
• each of C1-Cn is CR6R7, CHR6CHR7or CR6R7CH2, wherein R6 is hydrogen and R7 is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R6 and R7 are independently selected from the group consisting of hydrogen, (C2-C6) alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C1-C6) alkoxy, (C1-C6) alkylthio, NR3R4 and SR5, where R3 and R4 are as defined above, and R5 is hydrogen, (C1-C6) alkyl, hydroxy-, alkoxy-, or alkylthio- substituted (C1-C6) alkyl, or R6 and R7 taken together complete an alicyclic or heterocyclic system;
• each of D1-Dn is CR6R7, CH2CR6R7 or CHR6CHR7, where R6 and R7 are as defined above;
• each of G1-Gn-1 is
• in either orientation, wherein R3 is as defined above;
• Q is —CO2H, —CONR′R″, —SO3H or SO2NR′R″ or an activated derivative of —CO2H, —SO3H;
• 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, wherein C and D can optionally be CHR6(CH2)sssCHR7 where sss is from 0 to 2.

A method for sequencing a nucleic acid, said method comprising:

• contacting a nucleic acid-containing sample with a basically planar solid carrier comprising at least four single stranded peptide nucleic acid probes of different base sequence, each probe being covalently bound to a separate and distinct region of the basically planar solid carrier, and wherein at least one of the peptide nucleic acid probes each bind to different nucleic acids in said sample under conditions where the nucleic acid binds to the peptide nucleic acid probes and wherein the number of peptide nucleic acid probes bound to the solid carrier is equal to 4N, wherein N is the number of bases in each nucleic acid analogue;
• and
• determining the region of the carrier to which the nucleic acid binds as an indication of the sequence of the nucleic acid wherein the probes are nucleic acid analogs that have the general formula (I):
• wherein:
• n is at least 2,
• each of L1-Ln is independently selected from the group consisting of hydrogen, hydroxy, (C1-C4) alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups and reporter ligands, at least one of L1-Ln being a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, or a nucleobase-binding group;
• each of A1 through An is a single bond, a methylene group or a group of formula (IIa) or (IIb):
• wherein in (IIa) or (IIb):
• X is O, S, Se, NR3, CH2 or C(CH3)2;
• Y is a single bond, O, S or NR4;
• 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 ss is zero or an integer from 1 to 5, the sum r+ss being not more than 10;
• each R1 and R2 is independently selected from the group consisting of hydrogen, (C1-C4) alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen;
• and
• each R3 and R4 is independently selected from the group consisting of hydrogen, (C1-C4) alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C1-C4)alkyl, hydroxy, alkoxy, alkylthio and amino;
• and wherein in formula (I);
• each of B1-Bn is N or R3N+, where R3 is as defined above;
• each of C1-Cn is CR6R7, CHR6CHR7or CR6R7CH2, wherein R6 is hydrogen and R7 is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R6 and R7 are independently selected from the group consisting of hydrogen, (C2-C6) alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C1-C6) alkoxy, (C1-C6) alkylthio, NR3R4 and SR5, where R3 and R4 are as defined above, and R5 is hydrogen, (C1-C6) alkyl, hydroxy-, alkoxy-, or alkylthio- substituted (C1-C6) alkyl, or R6 and R7 taken together complete an alicyclic or heterocyclic system;
• each of D1-Dn is CR6R7, CH2CR6R7 or CHR6CHR7, where R6 and R7 are as defined above;
• each of G1-Gn-1 is
• in either orientation, wherein R3 is as defined above;
• Q is —CO2H, —CONR′R″, —SO3H or SO2NR′R″ or an activated derivative of —CO2H, —SO3H;
• 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, wherein C and D can optionally be CHR6(CH2)sssCHR7 where sss is from 0 to 2.

A method for the quantitative determination of a nucleic acid, said method comprising:

• contacting a sample that contains a target nucleic acid to be determined with a solid carrier comprising at least two single stranded peptide nucleic acid probes of different base sequence, each probe being covalently bound to a separate and distinct region of the solid carrier, and wherein at least one peptide nucleic acid probe binds to nucleic acid in said sample under conditions where the target nucleic acid binds to the peptide nucleic acid probe, and wherein at least one of said peptide nucleic acid probes has a base sequence that is complementary to the base sequence of the target nucleic acid, and wherein one additional peptide nucleic acid acid probe has a base sequence that is not complementary to the base sequence of the target nucleic acid, and wherein said target nucleic acid is detectably labeled;
• and
• determining the amount of target nucleic acid in said sample by determining the intensity of the detectable label bound in a region of the solid carrier that contains peptide nucleic acid probes having a base sequence that is complementary to the base sequence of the target nucleic acid wherein the probes are nucleic acid analogs that have the general formula (I):
• wherein:
• n is at least 2,
• each of L1-Ln is independently selected from the group consisting of hydrogen, hydroxy, (C1-C4) alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups and reporter ligands, at least one of L1-Ln being a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, or a nucleobase-binding group;
• each of A1 through An is a single bond, a methylene group or a group of formula (IIa) or (IIb):
• wherein in (IIa) or (IIb):
• X is O, S, Se, NR3, CH2 or C(CH3)2;
• Y is a single bond, O, S or NR4;
• 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 ss is zero or an integer from 1 to 5, the sum r+ss being not more than 10;
• each R1 and R2 is independently selected from the group consisting of hydrogen, (C1-C4) alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen;
• and
• each R3 and R4 is independently selected from the group consisting of hydrogen, (C1-C4) alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C1-C4)alkyl, hydroxy, alkoxy, alkylthio and amino;
• and wherein in formula (I);
• each of B1-Bn is N or R3N+, where R3 is as defined above;
• each of C1-Cn is CR6R7, CHR6CHR7or CR6R7CH2, wherein R6 is hydrogen and R7 is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R6 and R7 are independently selected from the group consisting of hydrogen, (C2-C6) alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C1-C6) alkoxy, (C1-C6) alkylthio, NR3R4 and SR5, where R3 and R4 are as defined above, and R5 is hydrogen, (C1-C6) alkyl, hydroxy-, alkoxy-, or alkylthio- substituted (C1-C6) alkyl, or R6 and R7 taken together complete an alicyclic or heterocyclic system;
• each of D1-Dn is CR6R7, CH2CR6R7 or CHR6CHR7, where R6 and R7 are as defined above;
• each of G1-Gn-1 is
• in either orientation, wherein R3 is as defined above;
• Q is —CO2H, —CONR′R″, —SO3H or SO2NR′R″ or an activated derivative of —CO2H, —SO3H;
• 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, wherein C and D can optionally be CHR6(CH2)sssCHR7 where sss is from 0 to 2.

A method for determining the location of two or more different peptide nucleic acid/nucleic acid complexes on a basically planar solid carrier, said method comprising:

• treating the carrier with an agent that deposits selectively on the complexes;
• and
• determining the location of the agent on said carrier to thereby determine the location of the two or more complexes on said carrier wherein the peptide nucleic acids are nucleic acid analogs that have the general formula (I):
• wherein:
• n is at least 2,
• each of L1-Ln is independently selected from the group consisting of hydrogen, hydroxy, (C1-C4) alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups and reporter ligands, at least one of L1-Ln being a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, or a nucleobase-binding group;
• each of A1 through An is a single bond, a methylene group or a group of formula (IIa) or (IIb):
• wherein in (IIa) or (IIb):
• X is O, S, Se, NR3, CH2 or C(CH3)2;
• Y is a single bond, O, S or NR4;
• 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 ss is zero or an integer from 1 to 5, the sum r+ss being not more than 10;
• each R1 and R2 is independently selected from the group consisting of hydrogen, (C1-C4) alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen;
• and
• each R3 and R4 is independently selected from the group consisting of hydrogen, (C1-C4) alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C1-C4)alkyl, hydroxy, alkoxy, alkylthio and amino;
• and wherein in formula (I);
• each of B1-Bn is N or R3N+, where R3 is as defined above;
• each of C1-Cn is CR6R7, CHR6CHR7or CR6R7CH2, wherein R6 is hydrogen and R7 is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R6 and R7 are independently selected from the group consisting of hydrogen, (C2-C6) alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C1-C6) alkoxy, (C1-C6) alkylthio, NR3R4 and SR5, where R3 and R4 are as defined above, and R5 is hydrogen, (C1-C6) alkyl, hydroxy-, alkoxy-, or alkylthio- substituted (C1-C6) alkyl, or R6 and R7 taken together complete an alicyclic or heterocyclic system;
• each of D1-Dn is CR6R7, CH2CR6R7 or CHR6CHR7, where R6 and R7 are as defined above;
• each of G1-Gn-1 is
• in either orientation, wherein R3 is as defined above;
• Q is —CO2H, —CONR′R″, —SO3H or SO2NR′R″ or an activated derivative of —CO2H, —SO3H;
• 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, wherein C and D can optionally be CHR6(CH2)sssCHR7 where sss is from 0 to 2.

The method of claim 64, wherein said peptide nucleic acids have the general formula (III):

• wherein:
• each L is independently selected from the group consisting of hydrogen, phenyl, heterocycles, one, two or three rings, naturally occurring nucleobases, and non-naturally occurring nucleobases;
• each R7′ 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 of k, 1 and m is independently zero or an integer from 1 to 5;
• and optionally the sum of k and m is 1 or 2;
• Rh is OH, NH2 or —NHLysNH2;
• and
• Ri is H or COCH3.