Modified labeled nucleotides and polynucleotides and methods of preparing, utilizing and detecting same

Claim 2

1 . A nucleotide having the general formula P-S-B-Sig wherein P is the phosphoric acid moiety, S the sugar or monosaccharide moiety, B being the base moiety, the phosphoric acid moiety being attached at the 3' and/or the 5' position of the sugar moiety when said nucleotide is a deoxyribonucleotide and at the 2', 3' and/or 5' position when said nucleotide is a ribonucleotide, said base being a purine or a pyrimidine, said base being attached from the N1 position or the N9 position to the 1' position of the sugar moiety when said base is a pyrimidine or a purine, respectively, and wherein said Sig is a chemical moiety covalently attached to the base B of said nucleotide, said Sig when attached to said base B being capable of signalling itself or makes itself self-detecting or its presence known.

Claim 3

A nucleotide in accordance with Claim 1 wherein Sig is attached to B at a position such that said nucleotide is capable of being incorporated into or to form a double-stranded ribonucleic acid, a double-stranded deoxyribonucleid acid or a double-stranded deoxyribonucleic acid-ribonucleic acid hybrid.

Claim 4

A nucleotide in accordance with Claim 1 wherein said Sig chemical moiety is a sugar selected from the group consisting of triose or tetrose, or pentose, a hexose, or heptose, and an octose.

Claim 5

A nucleotide in accordance with Claim 1 wherein said Sig chemical moiety comprises a component selected from the group consisting of an electron dense component, a magnetic component, an enzyme, a hormone component, a radioactive component, a metal-containing component, a fluorescing component and an antigen or antibody component.

Claim 6

A nucleotide in accordance with Claim I wherein said Sig chemical moiety is a sugar residue and wherein said sugar is complexed with or attached to a sugar or polysaccharide binding protein.

Claim 7

A nucleotide in accordance with Claim 1 wherein said Sig chemical moiety is connected to said base B via a chemical linkage.

Claim 8

A nucleotide in accordance with Claim 6 wherein said chemical linkage includes an olefinic bond at the a-position relative to base B.

Claim 9

A nucleotide in accordance with Claim 6 wherein said chemical linkage includes the moiety,

Claim 10

A nucleotide in accordance with Claim 1 wherein said Sig chemical moiety includes or comprises a catalytic metal component.

Claim 11

A single-stranded or a double-stranded polynucleotide comprising one or more nucleotides in accordance with Claim 1.

Claim 12

11 . A ribonucleotide having the general formula, . wherein P is the phosphoric acid moiety, S the sugar moiety and B the base moiety, the phosphoric acid moiety being attached at the 2', 3' and/or 5' position of the sugar moiety, said base B being attached from the N l position or the N9 position to the 1' position of the sugar moiety when said base is a pyrimidine or a purine, respectively, and wherein said Sig is a chemical moiety covalently attached to the sugar S, said Sig, when attached to'said sugar S, being capable of signalling itself or making itself self-detecting or its presence known.

Claim 13

11 . A ribonucleotide having the general formula, . wherein P is the phosphoric acid moiety, S the sugar moiety and B the base moiety, the phosphoric acid moiety being attached at the 2', 3' and/or 5' position of the sugar moiety, said base B being attached from the N l position or the N9 position to the 1' position of the sugar moiety when said base is a pyrimidine or a purine, respectively, and wherein said Sig is a chemical moiety covalently attached to the sugar S, said Sig, when attached to'said sugar S, being capable of signalling itself or making itself self-detecting or its presence known.

Claim 14

A ribonucleotide in accordance with Claim 11 wherein said Sig chemical moiety includes or comprises an enzyme.

Claim 15

A polyribonucleotide coupled or attached to a polypeptide.

Claim 16

A polyribonucleotide coupled or attached to a polypeptide.

Claim 17

A nucleotide having the general formula wherein P is the phosphoric acid moiety, S the sugar moiety and B the base moiety, the phosphoric acid moiety being attached to the 3' and/or the 5' position of the sugar moiety when said nucleotide is a deoxyribonucleotide and at the 2', 3' and/or 5' position when said nucleotide is a ribonucleotide, said base B being a purine or pyrimidine, said base B being attached from the Nl position or the N9 position to the 1' position of the sugar moiety when said base B is a pyrimidine or a purine, respectively, and wherein Sig is a chemical moiety covalently attached to the phosphoric acid moiety via the chemical linkage said Sig, when attached to said phosphoric acid moiety P being capable of signalling itself or making itself self-detecting or its presence known.

Claim 18

A nucleotide having the general formula wherein P is the phosphoric acid moiety, S the sugar moiety and B the base moiety, the phosphoric acid moiety being attached to the 3' and/or the 5' position of the sugar moiety when said nucleotide is a deoxyribonucleotide and at the 2', 3' and/or 5' position when said nucleotide is a ribonucleotide, said base B being a purine or pyrimidine, said base B being attached from the Nl position or the N9 position to the 1' position of the sugar moiety when said base B is a pyrimidine or a purine, respectively, and wherein Sig is a chemical moiety covalently attached to the phosphoric acid moiety via the chemical linkage said Sig, when attached to said phosphoric acid moiety P being capable of signalling itself or making itself self-detecting or its presence known.

Claim 19

A nucleotide having the general formula wherein P is the phosphoric acid moiety, S the sugar and monosaccharide moiety, B being the base moiety, the phosphoric acid moiety being attached to the 3' and/or the 5' position of the sugar moiety when said nucleotide is deoxyribonucleotide and at the 2', 3' and/or 5' position when said nucleotide is a ribonucleotide, said base being a purine or a pyrimidine, said base being attached from the Nl position or the N9 position to the Cl' position of the sugar moiety when said base is a pyrimidine or a purine, respectively, and wherein said Sig is a chemical moiety covalently attached to the base B of said nucleotide, said Sig being attached to the N 6 or 6-amino group when said base B is adenine or the N 2 or 2-amino group when said base B is guanine or the N 4 or 4-amino group when said base B is cytosine, said Sig when attached to said base B being capable of signally itself or makes itself self-detecting or its presence known.

Claim 20

A nucleotide having the general formula wherein P is the phosphoric acid moiety, S the sugar and monosaccharide moiety, B being the base moiety, the phosphoric acid moiety being attached to the 3' and/or the 5' position of the sugar moiety when said nucleotide is deoxyribonucleotide and at the 2', 3' and/or 5' position when said nucleotide is a ribonucleotide, said base being a purine or a pyrimidine, said base being attached from the Nl position or the N9 position to the Cl' position of the sugar moiety when said base is a pyrimidine or a purine, respectively, and wherein said Sig is a chemical moiety covalently attached to the base B of said nucleotide, said Sig being attached to the N 6 or 6-amino group when said base B is adenine or the N 2 or 2-amino group when said base B is guanine or the N 4 or 4-amino group when said base B is cytosine, said Sig when attached to said base B being capable of signally itself or makes itself self-detecting or its presence known.

Claim 21

A nucleotide having the general formula P-S-B, wherein P is the phosphoric acid moiety, S the sugar or monosaccharide moiety and B the base moiety, said nucleotide having covalently attached to the P or S or B moiety a chemical moiety Sig, said Sig chemical moiety when attached to the P moiety is attached thereto via the chemical linkage, and when Sig is attached to the S moiety, the S moiety is a ribose group, said chemical moiety Sig when attached to said P, S or B being capable of signalling itself or makes itself self-detecting or its presence known.

Claim 22

A nucleotide having the general formula P-S-B, wherein P is the phosphoric acid moiety, S the sugar or monosaccharide moiety and B the base moiety, said nucleotide having covalently attached to the P or S or B moiety a chemical moiety Sig, said Sig chemical moiety when attached to the P moiety is attached thereto via the chemical linkage, and when Sig is attached to the S moiety, the S moiety is a ribose group, said chemical moiety Sig when attached to said P, S or B being capable of signalling itself or makes itself self-detecting or its presence known.

Claim 23

A polynucleotide comprising one or more nucleotides in accordance with Claim 1 or Claim 11 or Claim 14 or Claim 15 or Claim 16, coupled or attached to a polypeptide, said polypeptide having attached thereto one or more Sig chemical moieties, said Sig when attached to said polypeptide being capable of signalling itself or making itself self-detecting or its presence known.

Claim 24

A nucleotide in accordance with Claim 1 or Claim 11 or Claim 14 or Claim 15 or Claim 16 wherein said Sig chemical moiety includes a chelating agent.

Claim 25

A nucleotide in accordance with Claim 1 or Claim 11 or Claim 14 or Claim 15 or Claim 16 wherein the Sig chemical moiety comprises the chemical moiety

Claim 28

A nucledtide in accordance with Claim 1, 11, 14, 15 or 16 wherein said Sig chemical moiety includes a coenzyme.

Claim 29

A polynucleotide containing one or more nucleotides in accordance with Claim 1, or Claim 11, or Claim 14, or Claim 15, or Claim 16 wherein said Sig moiety is radio- )actively labeled.

Claim 30

A polynucleotide containing one or more nucleotides in accordance with Claims 11,14,15 or 16 wherein said Sig chemical moiety comprises biotin attached to radioactively 5labeled avidin, or to radioactively labeled strepavidin.

Claim 31

A polynucleotide containing one or more nucleotides, in accordance with Claim 1 or Claim 11, or Claim 14, or Claim 15, or Claim 16, wherein said Sig moiety comprises a radioactively labeled antibody or a radioactively labeled protein.

Claim 32

A method for the stimulation or induction of cells for the production of lymphokines, cytokinins and/or interferon which comprises introducing into or bringing into contact with cells capable of and/or functioning for the production of said lymphokines, cytokinins and/or interferon an effective lymphokine, cytokinin and/or interferon stimulating and production inducing amount of a nucleotide in accordance with Claim 1, 11, 14, 15, or 16.

Claim 33

The compound wherein M is hydrogen or a metal.

Claim 34

The compound wherein M is hydrogen or a metal.

Claim 35

The compound wherein M is hydrogen or a metal.

Claim 36

The compound wherein M is hydrogen or a metal.

Claim 37

'27. The compound wherein M is hydrogen or a metal.

Claim 38

'27. The compound wherein M is hydrogen or a metal.

Claim 39

An amino acid or polypeptide comprising a Sig moiety attached thereto.

Claim 40

An amino acid or polypeptide comprising a Sig moiety attached thereto.

Claim 41

A monosaccharide or a polysaccharide comprising a Sig moiety attached thereto.

Claim 42

A monosaccharide or a polysaccharide comprising a Sig moiety attached thereto.

Claim 43

A method of detecting a first compound which includes a nucleotide in accordance with Claim 1, 11, 14, 15 or 16 as part of said first compound, which comprises contacting said first compound with a second compound capable of forming a complex therewith under suitable conditions so as to form said complex, said complex comrising said first compound and said second compound and detecting said complex.

Claim 44

A method of determining the presence of a deoxyribonucleic or ribonucleic acid molecule which comprises forming a double-stranded hybrid polynucleotide duplex which includes a single strand of deoxyribonucleic acid or ribonucleic acid corresponding to or derived from said deoxyribonucleic or ribonucleic acid molecule and a nucleotide in accordance with Claim 1, 11, 14, 15 or 16 and detecting said duplex.

Claim 45

A method of testing a bacterium to determine its resistance to an antibiotic which comprises preparing a polynucleotide complementary to the deoxyribonucleic acid gene sequence of said bacterium which confers resistance of said bacterium to said antibiotic and which includes a nucleotide in accordance with Claim 1,11,14,15 or 16 incorporated therein, contacting said polynucleotide under suitable conditions with a deoxyribonucleic acid obtained from said bacterium so as to form a double-stranded hybrid duplex and detecting the presence of said duplex, the detection of said duplex indicating resistance of said bacterium to said antibiotic and the absence of said duplex indicating the susceptibility of said bacterium to an antibiotic. --

Claim 46

A method of diagnosing a genetic disorder in a subject which comprises preparing a polynucleotide complementary to the deoxyribonucleic acid gene sequence of said subject which is associated with said genetic disorder and includes a compound in accordance with Claim 1,11,14 ,15 or 16, incorporated therein contacting said polynucleotide under suitable conditions with deoxyribonucleic acid obtained from said subject so as to form a double-stranded hybrid duplex and detecting the presence of said hybrid duplex, the presence or absence of said hybrid duplex indicating the presence or absence of said genetic disorder. --

Claim 47

A method of diagnosing thalassemia which comprises preparing a polynucleotide complementary to the deoxyribonucleic acid gene sequence which is absent in B-minus-thalassemia subjects and includes a nucleotide in accordance with Claim 1, 11, 14, 15 or 16, contacting said polynucleotide under suitable conditions with deoxyribonucleic acid obtained from said subject so as to form a double-stranded hybrid duplex and determining the presence of said duplex, the absence of said duplex indicating the presence of B-minus-thalassemia.

Claim 48

A method of chromosomal karyotyping which comprises preparing a series of modified polynucleotides corresponding to a series of defined genetic sequences located on chromosomes, said polynucleotides including one or more compounds in accordance with Claim 1, 11, 14, 15 or 16, contacting said polynucleotides with deoxyribonucleic acid of or obtained from chromosomes so as to form hybrid duplexes and detecting said duplexes, thereby determining the location of said duplexes on said chromosomes and the location of said genetic sequences on said chromosomes.

Claim 49

A method of diagnosing a tumor cell which comprises preparing a polynucleotide which is complementary to a messenger ribonucleic acid synthesized from a deoxyribonucleic acid gene sequence associated with the production of a polypeptide diagnostic for or identifiable with said tumor cell or which is complementary to said deoxyribonucleic acid gene sequence and which includes a compound in accordance with Claim 1, 11, 14, 15 or 16 introducing said polynucleotide into contact with said cell under suitable conditions so as to permit said polynucleotide to hybridize with said deoxyribonucleic acid gene sequence or said messenger-ribonucleic acid and detecting the formation of a resulting formed hybrid containing said polynucleotide.

Claim 56

40 .A method of determining or identifying or diagnosing in an organism, cellular material or tissue a first compound comprising a receptor molecule with a receptor site that specifically binds to a second molecule which comprises bringing said organism, material or tissue containing said receptor molecule into contact with a second compound, said second compound comprising a first component capable of complexing with or binding with said receptor molecule, said second compound also comprising a second component, said second component being attached to or complexed with said first component, said first component of said second compound when in contact with said receptor molecule complexes with said receptor molecule to form a third compound comprising said first compound and said second compound and contacting said third compound with a fourth compound capable of binding with or attaching to said second component of said second compound making.up said third compound, said first component of said second compound being selected from group A consisting of an amino acid, a peptide, or a protein or from group B consisting of a sugar, oligosaccharide or polysaccharide or from group C consisting of a purine, pyrimidine, nucleoside, nucleotide, oligonucleotide or polynucleotide, said second component of said second compound being a sugar, oligosaccharide or polysaccharide and said fourth compound is selected from the group consisting of a peptide or polypeptide, a lectin, or an antibody. --

Claim 57

40 .A method of determining or identifying or diagnosing in an organism, cellular material or tissue a first compound comprising a receptor molecule with a receptor site that specifically binds to a second molecule which comprises bringing said organism, material or tissue containing said receptor molecule into contact with a second compound, said second compound comprising a first component capable of complexing with or binding with said receptor molecule, said second compound also comprising a second component, said second component being attached to or complexed with said first component, said first component of said second compound when in contact with said receptor molecule complexes with said receptor molecule to form a third compound comprising said first compound and said second compound and contacting said third compound with a fourth compound capable of binding with or attaching to said second component of said second compound making.up said third compound, said first component of said second compound being selected from group A consisting of an amino acid, a peptide, or a protein or from group B consisting of a sugar, oligosaccharide or polysaccharide or from group C consisting of a purine, pyrimidine, nucleoside, nucleotide, oligonucleotide or polynucleotide, said second component of said second compound being a sugar, oligosaccharide or polysaccharide and said fourth compound is selected from the group consisting of a peptide or polypeptide, a lectin, or an antibody. --

Claim 58

41 . A method of detecting a'first compound which includes in its make-up a nucleotide in accordance with Claim 20, which comprises contacting said first compound with an apoenzyme corresponding to said coenzyme. --

Claim 59

A method of determining the presence of a polynucleotide, comprising one or more nucleotides in accordance with Claim 1, 11,14,15 or 16, wherein said Sig component of said nucleotide comprises biotin which comprises bringing said polynucleotide into contact with radioactive labeled avidin or strepavidin to bind said biotin to said radioactive labeled avidin or strepavidin and determining the presence of the biotin bound to said radioactive labeled avidin or strepavidin by detecting the radioactivity of the biotin-bound radioactive avidin or strepavidin.

Claim 60

A diagnostic kit useful for determining the presence of a nucleic acid-containing organism or the like which comprises a polynucleotide which includes in its make-up a compund selected from the compounds of Claim 1, 11, 14, 15 or 16 and which is complementary to all or an identifiable, distinct or unique portion of the nucleic acid contained in said organism and means for detecting or expressing the presence or absence of a resulting formed hybrid between said polynucleotide and said nucleic acid of said organism when said polynucleotide is brought into contact with the nucleic acid of said organism or the like under hybrid forming conditions.