EP0063879A2
Expired / Lapsed
EP
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Modified nucleotides and methods of preparing and using same
Assignee: Yale University
•
Filing: 1982-04-06
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Granted: —
•
Expires: 2002-04-06
Abstract
Abstract Compounds having the structure: wherein B represents a purine, 7-deazapurine, or pyrimidine moiety covalently bonded to the C 1 -position of the sugar moiety, provided that when B is purine or 7-deazapurine. it is attached at the N'-position of the purine or 7-deazapurine and when 8 is pyrimidine, it is attached at the N'-position; wherein A represents a moiety consisting of at least three carbon atoms which is capable of forming a detectable complex with a polypeptide when the compound is Incorporated into a double-stranded ribonucleic acid. deoxyribonucleic acid duplex, or DNA-RNA hybrid; wherein the dotted line represents a chemical linkage joining B and A. provided that if B is purine, the linkage is attached to the 8-position of the purine, if B is 7. deazapurine, the linkage is attached to the 7-position of the deazapurine, and if B is pyrimidine, the linkage is attached to the 5-position of the pyrimidine; and wherein each of x. y and z represents either directly, or when incorporated into oligo- and polynucleotides, provide probes which are widely useful. Applications include detection and localization of polynucleotide sequences in chromosomes, fixed cells. tissue sections, and cell extracts. Specific applications include chromosomal karyotyping, clinical diagnosis of nucleic acid-containing etiological agents, e.g. bacteria, viruses. or fungi, and diagnosis of genetic disorders.
Inventors
- 👤 David C. Ward
- 👤 Alexander A. Waldrop, Iii
- 👤 Pennina R. Langer
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-24 03:56
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/02; C12Q1/18; C07; C07H; C07H19/00; C07H19/02; C07H19/04; C07H19/16; C07H19/20; C07H21/00; C12P; C12P19/00; C12P19/26; C12P19/28; C12P19/30; C12P19/34; C12Q1/68; C12Q1/6804; C12Q1/6813; C12Q1/6816
Claims Analysis
Total Claims: 59
Claim 1
Independent Claim
165 words
A compound having the structure: wherein B represents a purine, 7-deazapurine or pyrimidine moiety covalently bonded to the C 1' -position of the sugar moiety, provided that when B is purine or 7-deazapurine, it is attached at the N 9 -position of the purine or deazapurine, and when B is pyrimidine, it is attached at the N l -position; wherein A represents a moiety consisting of at least three carbon atoms which is capable of forming a detectable complex with a polypeptide when the compound is incorporated into a double-stranded ribonucleic acid, deoxyribonucleic acid duplex, or DNA-RNA hybrid; wherein the dotted line represents a linkage or group joining B and A, provided that if B is purine, the linkage is attached to the 8-position of the purine, if B is 7-deazapurine, the linkage is attached to the 7-position of the deazapurine, and if B is pyrimidine, the linkage is attached to the 5-position of the pyrimidine; and wherein each of x, y and z represents
Claim 2
Dependent Claim
165 words
A compound having the structure: wherein B represents a purine, 7-deazapurine or pyrimidine moiety covalently bonded to the C 1' -position of the sugar moiety, provided that when B is purine or 7-deazapurine, it is attached at the N 9 -position of the purine or deazapurine, and when B is pyrimidine, it is attached at the N l -position; wherein A represents a moiety consisting of at least three carbon atoms which is capable of forming a detectable complex with a polypeptide when the compound is incorporated into a double-stranded ribonucleic acid, deoxyribonucleic acid duplex, or DNA-RNA hybrid; wherein the dotted line represents a linkage or group joining B and A, provided that if B is purine, the linkage is attached to the 8-position of the purine, if B is 7-deazapurine, the linkage is attached to the 7-position of the deazapurine, and if B is pyrimidine, the linkage is attached to the 5-position of the pyrimidine; and wherein each of x, y and z represents
Claim 3
Dependent Claim
189 words
A compound having the structure: wherein each of B, B', and B" represents a purine, deazapurine, or pyrimidine moiety covalently bonded to the C 1' - position of the sugar moiety, provided that whenever B, B', or B" is purine or deazapurine, it is attached at the N9- position of the purine or deazapurine, and whenever B, B', or B" is pyrimidine, it is attached at the N 1 -position; wherein A represents a moiety consisting of at least three carbon atoms which is capable of forming a detectable complex with a. polypeptide when the compound is incorporated into a double-stranded duplex formed with a complementary ribonucleic or deoxyribonucleic acid molecule. wherein the dotted line represents a chemical linkage or group joining B and A, provided that if B is purino, tho linkage is attached to the 8-position of the purine, if B is 7-deazapurine, the linkage is attached to the 7-position of the deazapurine, and if B is pyrimidine, the linkage is attached to the 5-position of the pyrimidine; wherein z represents H- or HO-; and wherein m and h represent integers from 0 up to about 100,000.
Claim 4
Dependent Claim
189 words
A compound having the structure: wherein each of B, B', and B" represents a purine, deazapurine, or pyrimidine moiety covalently bonded to the C 1' - position of the sugar moiety, provided that whenever B, B', or B" is purine or deazapurine, it is attached at the N9- position of the purine or deazapurine, and whenever B, B', or B" is pyrimidine, it is attached at the N 1 -position; wherein A represents a moiety consisting of at least three carbon atoms which is capable of forming a detectable complex with a. polypeptide when the compound is incorporated into a double-stranded duplex formed with a complementary ribonucleic or deoxyribonucleic acid molecule. wherein the dotted line represents a chemical linkage or group joining B and A, provided that if B is purino, tho linkage is attached to the 8-position of the purine, if B is 7-deazapurine, the linkage is attached to the 7-position of the deazapurine, and if B is pyrimidine, the linkage is attached to the 5-position of the pyrimidine; wherein z represents H- or HO-; and wherein m and h represent integers from 0 up to about 100,000.
Claim 5
Dependent Claim
14 words
A compound in accordance with Claim 1 or 2 wherein A is a hapten.
Claim 6
Dependent Claim
14 words
A compound in accordance with Claim 1 or 2 wherein A is a ligand.
Claim 7
Dependent Claim
13 words
A compound in accordance with Claim 1 or 2 wherein A is biotin.
Claim 8
Dependent Claim
14 words
. A compound in accordance with Claim 1 or 2 wherein A is iminobiotin.
Claim 9
Dependent Claim
19 words
A compound in accordance with Claim 1 wherein A is an organic moiety containing at least five carbon atoms.
Claim 10
Dependent Claim
14 words
A compound in accordance with Claim 1 wherein A is a non-aromatic organic moiety.
Claim 11
Dependent Claim
28 words
A compound in accordance with Claim 1 or 2 wherein the chemical linkage represented by the dotted line includes an olefinic bond at the α-position relative to B.
Claim 12
Dependent Claim
18 words
A compound in accordance with Claim 1 or 2 wherein the chemical.linkage includes the moiety -CH 2 -NH-.
Claim 13
Dependent Claim
17 words
A compound in accordance with Claim 9 wherein the ole- .finic chemical linkage is -CH=CH-CH 2 -NH-.
Claim 14
Dependent Claim
13 words
A compound in accordance with Claim 9 wherein the olefinic chemical linkage is
Claim 15
Dependent Claim
24 words
A compound in accordance with Claim 1 wherein the chemical linkage is selected from or includes a moiety selected from the group consisting of
Claim 16
Dependent Claim
15 words
A compound in accordance with Claim 1 wherein B is uracil, cytosine, deazaadenine, or deazaguanine.
Claim 17
Dependent Claim
17 words
A compound in accordance with Claim 1 wherein x is y is HO-, and z is HO-
Claim 18
Dependent Claim
17 words
A compound in accordance with Claim 1 wherein x is y is HO-, and z is HO-.
Claim 19
Dependent Claim
17 words
A compound in accordance with Claim 1 wherein x is y. is HO-, and z is HO-.
Claim 20
Dependent Claim
17 words
A compound in accordance with Claim 1 wherein x is y is HO-, and z is H-.
Claim 21
Dependent Claim
16 words
A compound in accordance with Claim 1 wherein x.is y is HO-, and z is H-.
Claim 22
Dependent Claim
17 words
A compound in accordance with Claim 1 wherein x is y is HO-, and z is H-.
Claim 23
Dependent Claim
17 words
A compound in accordance with Claim 1 wherein x is , y is and z is HO-.
Claim 24
Dependent Claim
16 words
A compound in accordance with Claim 1 wherein x is y is and z is HO-.
Claim 25
Dependent Claim
15 words
A compound in accordance with any of Claims 15 to 22, wherein A is biotin.
Claim 26
Dependent Claim
15 words
A compound in accordance with any of Claims 15 to 22 wherein A is iminobiotin.
Claim 27
Dependent Claim
19 words
A compound in accordance with any of Claims 15 to 24 wherein the chemical linkage is -CH=CH-CH 2 -NH-.
Claim 28
Dependent Claim
16 words
A compound in accordance with any of Claims 15 to 24 wherein the chemical linkage is
Claim 29
Dependent Claim
15 words
A compound in accordance with Claim 2 wherein m and n are not simultaneously 0.
Claim 30
Dependent Claim
15 words
A compound in accordance with Claim 2 wherein B is uracil, cytosine, deazaadenine, or deazaguanine.
Claim 31
Independent Claim
321 words
A method of preparing a modified nucleotide having the structure: wherein B represents a purine, 7-deazapurine or pyrimidine moiety covalently bonded to the C 1' -position of the sugar moiety, provided that when B'is purine or 7-deazapurine, it is attached at the N 9 -position of the purine or deazapuring, and when B is pyrimidine, it is attached at the N 1 -position; wherein A represents a moiety consisting of at least three carbon atoms which is capable of forming a detectable complex with a polypeptide when the compound is incorporated into a double-stranded ribonucleic acid, deoxyribonucleic acid duplex, DNA-RNA hybrid; wherein the dotted line represents a chemical linkage joining B and A, provided that if B is purine, the linkage is attached to the 8-position of the purine, if B is 7-deazapurine, the linkage is attached to the 7-position of the deazapurine, and if B is pyrimidine, the linkage is attached to the 5-position of the pyrimidine; and wherein each of.z, y, and z represents comprising the steps of: (a) reacting a compound having the structure: with a mercuric salt in a suitable solvent under suitable conditions so as to form a mercurated compound having the structure: (b) reacting said mercurated compound with a chemical moiety reactive with the -Hg + portion of said mercurated compound and represented by the formula ···N, said reaction being carried out in'an aqueous solvent and in the presence of K 2 PdCl 4 under suitable conditions so as to form a compound having the structure: wherein N is a reactive terminal functional group or is A; and (c) recovering said compound as said modified nucleotide when N is A, or when N is a reactive terminal group, reacting said compound with a compound having the structure M-A, wherein M represents a functional group reactive with N in an aqueous solvent under suitable conditions so as to form said modified nucleotide which is then recovered.
Claim 32
Independent Claim
321 words
A method of preparing a modified nucleotide having the structure: wherein B represents a purine, 7-deazapurine or pyrimidine moiety covalently bonded to the C 1' -position of the sugar moiety, provided that when B'is purine or 7-deazapurine, it is attached at the N 9 -position of the purine or deazapuring, and when B is pyrimidine, it is attached at the N 1 -position; wherein A represents a moiety consisting of at least three carbon atoms which is capable of forming a detectable complex with a polypeptide when the compound is incorporated into a double-stranded ribonucleic acid, deoxyribonucleic acid duplex, DNA-RNA hybrid; wherein the dotted line represents a chemical linkage joining B and A, provided that if B is purine, the linkage is attached to the 8-position of the purine, if B is 7-deazapurine, the linkage is attached to the 7-position of the deazapurine, and if B is pyrimidine, the linkage is attached to the 5-position of the pyrimidine; and wherein each of.z, y, and z represents comprising the steps of: (a) reacting a compound having the structure: with a mercuric salt in a suitable solvent under suitable conditions so as to form a mercurated compound having the structure: (b) reacting said mercurated compound with a chemical moiety reactive with the -Hg + portion of said mercurated compound and represented by the formula ···N, said reaction being carried out in'an aqueous solvent and in the presence of K 2 PdCl 4 under suitable conditions so as to form a compound having the structure: wherein N is a reactive terminal functional group or is A; and (c) recovering said compound as said modified nucleotide when N is A, or when N is a reactive terminal group, reacting said compound with a compound having the structure M-A, wherein M represents a functional group reactive with N in an aqueous solvent under suitable conditions so as to form said modified nucleotide which is then recovered.
Claim 33
Dependent Claim
16 words
A method.in accordance with Claim 25 wherein said chemical moiety represented by the formula ···N is
Claim 34
Dependent Claim
16 words
A method in accordance with Claim 25 wherein said chemical moiety.represented by the formula ···N is
Claim 35
Dependent Claim
64 words
A method of making a compound in accordance with Claim 2 comprising an enzymatically polymerization of nucleotide triphosphates having the structure wherein Q represents B···A, B', or B", and one of x and y represents , and the other of x and y represents HO- in the presence of a nucleic acid template under suitable conditions so as to form said com- . pound.
Claim 36
Dependent Claim
171 words
A method of making a compound in accordance with Claim 2 comprising the steps of: (a) reacting a compound having the structure: with a mercuric salt in a suitable solvent under suitable conditions so as to form a mercurated derivative compound having the structure: (b) reacting said mercurated derivative compound with a chemical moiety reactive with the -Hg + portion of said mercurated compound and represen- tod by the formula ···N, said reaction being carried out in an aqueous solvent and in the presence of K 2 PdCl 4 under suitable conditions so as to form a compound having the structure: wherein N is a reactive terminal functional group or is A; and (c) recovering said compound of Claim 2 when N is A, or when N is a reactive terminal group, reacting said compound with a compound having the structure M-A wherein M represents a functional group reactive with N in an aqueous solvent under suitable conditions so as to form said compound of Claim 2 which is then recovered.
Claim 37
Dependent Claim
16 words
A method in accordance with Claim 33 wherein ···N comprises thiol, carboxylic acid, epoxide, and amine.
Claim 38
Dependent Claim
17 words
A method in accordance with Claim 33 wherein said chemical moiety represented by the formula ···N is
Claim 39
Dependent Claim
17 words
A method in accordance with Claim 33 wherein said chemical moiety represented by the formula ···N is
Claim 40
Dependent Claim
17 words
A method in accordance with Claim 33 wherein said chemical moiety represented by the formula ···N is
Claim 41
Independent Claim
16 words
A method in accordance with Claim'33 wherein said chemical moiety represented by the formula ···N is
Claim 42
Dependent Claim
67 words
A method of detecting a compound in accordance with Claim 1 which comprises contacting said compound with a polypeptide capable of forming a complex therewith under suitable conditions so as to form said complex, said polypepetide being capable of or including a moiety which can be detected when said complex of said compound and said polypeptide is formed, and detecting said complex using an appropriate detection technique.
Claim 43
Dependent Claim
22 words
A chemical complex comprising a compound in accordance with Claim 1 and a polypeptide capable of forming said complex with said compound.
Claim 44
Dependent Claim
63 words
A method of detecting a compound in accordance with Claim 2 which comprises contacting said compound with a polypeptide capable of forming a complex therewith under suitable conditions so as to form said complex, said polypeptide including a moiety which can be detected when said complex of said compound and said polypeptide is formed, and detecting said complex using an appropriate detection technique.
Claim 45
Dependent Claim
22 words
A chemical complex comprising a compound in accordance with Claim 2 and a polypeptide capable of forming said complex with said compound.
Claim 46
Dependent Claim
34 words
A chemical complex in accordance with Claim 42 wherein said polypeptide includes a moiety which can be detected, being a fluorescent dye, electron dense reagent, or enzyme capable of depositing an insoluble reaction product.
Claim 47
Dependent Claim
70 words
A method of detecting a double-stranded polynucleotide duplex which includes a compound in accordance with either Claim 1 or Claim 2 which comprises contacting said polynucleotide duplex with a polypeptide capable of forming a complex therewith under suitable conditions so as to form said complex, said polypeptide including a moiety which can be detected when said complex of said polynucleotide duplex and said polypeptide is formed, and detecting said complex.
Claim 48
Dependent Claim
17 words
A double-stranded polynucleotide duplex which includes a compound in accordance with either Claim 1 or Claim 2.
Claim 49
Dependent Claim
63 words
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 or ribonucleic acid corresponding to or derived from said deoxyribonucleic or ribonucleic acid molecule and a compound in accordance with Claim 2, and detecting said double-stranded hybrid polynucleotide duplex according to the method of Claim 44.
Claim 50
Dependent Claim
101 words
A method of diagnosing the presence of a nucleic acid-containing etiological agent in a subject which comprises obtaining a suitable sample from said subject, determining the presence in said sample of deoxyribonucleic or ribonucleic acid naturally associated with said etiological agent by forming a double-stranded polynucleotide duplex which includes a compound in accordance with Claim 2 and a single strand of deoxyribonucleic or ribonucleic acid corresponding to or derived from said deoxyribonucleic or ribonucleic acid which is naturally associated with said etiological agent under suitable conditions, and detecting the presence of said double-stranded polynucleotide duplex using the method of Claim 44.
Claim 51
Dependent Claim
130 words
A method of testing a bacterium to determine the presence of resistance to an antibiotic which comprises preparing a polynucleotide complementary to the deoxyribonucleic acid gene sequence of said bacterium which confers resistance to said antibiotic and includes the compound of Claim 1 incorporated therein, contacting said polynucleotide with deoxyribonucleic acid obtained from said bacterium under suitable conditions so as to form a double-stranded hybrid duplex, contacting said duplex with a polypeptide capable of forming a complex with said hybrid duplex under suitable conditions, said polypeptide including a moiety which can be detected if said complex is formed, and detecting the presence of said complex using an appropriate detection technique, the'presence of said complex indicating resistance to said antibiotic and the absence of said complex indicating susceptibility to said antibiotic.
Claim 52
Dependent Claim
119 words
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 the compound of Claim 1 incorporated therein, contacting said polynucleotide with deoxyribonucleic acid obtained from said subject under suitable conditions so as to form a double-stranded hybrid duplex, contacting said duplex with a polypeptide capable of forming a complex with said hybrid duplex, said polypeptide including a moiety which can be detected when said complex is formed, and detecting the presence of said complex using an appropriate detection technique, the presence or absence of said complex indicating the presence or absence of said genetic disorder.
Claim 53
Dependent Claim
114 words
A method of diagnosing thalassemia in a human subject which comprises preparing a polynucleotide complementary to the deoxyribonucleic acid gene sequence which is absent in6 -minus thalassemia subjects and includes the compound of Claim 1 incorporated therein, contacting said polynucleotide with deoxyribonucleic acid obtained from said subject under suitable conditions so as to form a double-stranded hybrid duplex, contacting said duplex with a polypeptide capable of forming a complex with said hybrid duplex under suitable conditions, said polypeptide including a moiety which can be detected when said complex is formed, and detecting the presence of said complex using an appropriate detection technique, the absence of said complex indicating the presence of$ - minus thalassemia.
Claim 54
Dependent Claim
104 words
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 compounds in accordance with Claim 1, contacting said polynucleotides with deoxyriboxyribonucleic acid obtained from chromosomes so as to form hybrid duplexes, contacting each of said duplexes with a polypeptide which is capable of forming a complex with each such duplex, said polypeptides including moieties which can be detected when said complexes are formed, and determing the location of each complex on said chromosomes so as to thereby determine the location of said genetic sequences on said chromosomes.
Claim 55
Independent Claim
110 words
A method of detecting a polynucleotide which includes the terminal polynucleotide sequence poly A which comprises preparing a modified poly U molecule in which at least one uracil moiety has been modified by chemical addition at the 5-position of a moiety A consisting of at least three carbon atoms which is capable of forming a detectable complex with a polypeptide when the modified uracil moiety is incorporated into a double-stranded poly A-poly U duplex, forming such a poly A-poly U duplex by contacting said polynucleotide containing said poly A sequence with said modified poly U molecule under suita- conditions, and detecting resulting duplexes so as to thereby detect said polynucleotide.
Claim 56
Independent Claim
110 words
A method of detecting a polynucleotide which includes the terminal polynucleotide sequence poly A which comprises preparing a modified poly U molecule in which at least one uracil moiety has been modified by chemical addition at the 5-position of a moiety A consisting of at least three carbon atoms which is capable of forming a detectable complex with a polypeptide when the modified uracil moiety is incorporated into a double-stranded poly A-poly U duplex, forming such a poly A-poly U duplex by contacting said polynucleotide containing said poly A sequence with said modified poly U molecule under suita- conditions, and detecting resulting duplexes so as to thereby detect said polynucleotide.
Claim 57
Dependent Claim
102 words
A method of tumor or cancer cell identification which comprises detecting malignant cells by detecting abnormal hormonal receptor sites associated therewith by providing: (1) a compound in accordance with Claim 1 in which x or z is H- or HO- and the other two of x, y and z are reacted to form the cyclic moiety. (2) and binding said compound to the said sites under suitable conditions permitting binding, disrupting said cells to produce cell surface fragments to which said compound is bound, separately recovering said cell surface fragment, and identifying the same so as to identify said hormone receptor sites.
Claim 58
Dependent Claim
76 words
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 production of a polypeptide diagnostic for said tumor cell and includes a compound in accordance with Claim 1, introducing said polynucleotide into said cell under suitable conditions so as to permit said polynucleotide to hybridize with said deoxyribonucleic acid gene sequence, and determining whether said polynucleotide hybridizes.
Claim 59
Dependent Claim
55 words
A diagnostic kit useful for determining the presence of a nucleic acid - containing organism such as a bacterium which comprises a compound in accordance with Claim 2 which is complementary to all or a unique portion of the nucleic acid contained in said organism and a polypeptide capable of forming a detectable complex therewith.
References Cited (Backward Citations)
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