US5449767A
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Modified polynucleotides and methods of preparing same
Assignee: Yale University
•
Filing: 1992-05-20
•
Granted: 1995-09-12
•
Expires: 2012-05-20
Abstract
Abstract Compounds having the structure: <IMAGE> wherein B represents 7-deazapurine, or pyrimidine moiety covalently bonded to the C1'-position of the sugar moiety, provided that when B is 7-deazapurine, it is attached at the N9-position of the 7-deazapurine and when B is pyrimidine, it is attached at the N1-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 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 <IMAGE> or <IMAGE> either directly, or when incorporated into oligo- and polynucleotides, provide probes which are widely useful.
Inventors
- 👤 David C. Ward
- 👤 Pennina R. Langer
- 👤 Alexander A. Waldrop, III
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 13:48
CPC Classifications
C; C07; C07H; C07H19/00; C07H19/02; C07H19/04; C07H19/16; C07H19/20; C07H21/00; C12; C12P; C12P19/00; C12P19/26; C12P19/28; C12P19/30; C12P19/34; C12Q; C12Q1/00; C12Q1/02; C12Q1/18; C12Q1/68; C12Q1/6804; C12Q1/6813; C12Q1/6816
Claims Analysis
Total Claims: 75
Claim 1
Independent Claim
211 words
An oligo- or polynucleotide containing a nucleotide having the structure: ##STR26## wherein B represents a 7-deazapurine or a pyrimidine moiety covalently bonded to the C 1 '-position of the sugar moiety, provided that whenever B is a 7-deazapurine, the sugar moiety is attached at the N 9 -position of the 7-deazapurine, and whenever B is a pyrimidine, the sugar moiety is attached at the N 1 -position of the pyrimidine; wherein A comprises at least three carbon atoms and represents at least one component of a signaling moiety capable of producing a detectable signal; wherein B and A are covalently attached directly or through a linkage group that does not substantially interfere with the characteristic ability of the oligo- or polynucleotide to hybridize with a nucleic acid and does not substantially interfere with formation of the signalling moiety or detection of the detectable signal, provided also that if B is 7-deazapurine, A or the linkage group is attached to the 7-position of the deazapurine, and if B is pyrimidine, A or the linkage group is attached to the 5-position of the pyrimidine; wherein one of x and y represents ##STR27## and the other of x and y is absent or represents --OH or --H; and wherein z represents H-- or HO--.
Claim 2
Dependent Claim
211 words
An oligo- or polynucleotide containing a nucleotide having the structure: ##STR26## wherein B represents a 7-deazapurine or a pyrimidine moiety covalently bonded to the C 1 '-position of the sugar moiety, provided that whenever B is a 7-deazapurine, the sugar moiety is attached at the N 9 -position of the 7-deazapurine, and whenever B is a pyrimidine, the sugar moiety is attached at the N 1 -position of the pyrimidine; wherein A comprises at least three carbon atoms and represents at least one component of a signaling moiety capable of producing a detectable signal; wherein B and A are covalently attached directly or through a linkage group that does not substantially interfere with the characteristic ability of the oligo- or polynucleotide to hybridize with a nucleic acid and does not substantially interfere with formation of the signalling moiety or detection of the detectable signal, provided also that if B is 7-deazapurine, A or the linkage group is attached to the 7-position of the deazapurine, and if B is pyrimidine, A or the linkage group is attached to the 5-position of the pyrimidine; wherein one of x and y represents ##STR27## and the other of x and y is absent or represents --OH or --H; and wherein z represents H-- or HO--.
Claim 3
Dependent Claim
21 words
An oligo- or polynucleotide of claim 1 wherein B is selected from the group consisting of uracil, cytosine, deazaadenine, and deazaguanine.
Claim 4
Dependent Claim
12 words
An oligo- or polynucleotide of claim 1 wherein A is a ligand.
Claim 5
Dependent Claim
12 words
An oligo- or polynucleotide of claim 1 wherein A is a hapten.
Claim 6
Dependent Claim
11 words
An oligo- or polynucleotide of claim 4 wherein A is biotin.
Claim 7
Dependent Claim
11 words
An oligo- or polynucleotide of claim 4 wherein A is iminobiotin.
Claim 8
Dependent Claim
21 words
An oligo- or polynucleotide of claim 1 wherein the linkage group includes an olefinic bond at the α-position relative to B.
Claim 9
Dependent Claim
17 words
An oligo- or polynucleotide of claim 1 wherein the linkage group includes the moiety --CH 2 --NH--.
Claim 10
Dependent Claim
18 words
An oligo- or polynucleotide of claim 7 wherein the olefinic linkage group is the moiety --CH═CH--CH 2 --NH--.
Claim 11
Dependent Claim
16 words
An oligo- or polynucleotide of claim 7 wherein the wherein the olefinic linkage group is ##STR28##
Claim 12
Dependent Claim
19 words
An oligo- or polynucleotide of claim 1 wherein A is an organic moiety containing at least five carbon atoms.
Claim 13
Dependent Claim
11 words
An oligo- or polynucleotide of claim 1 wherein z is HO--.
Claim 14
Dependent Claim
11 words
An oligo- or polynucleotide of claim 1 wherein z is H--.
Claim 15
Dependent Claim
11 words
An oligo- or polynucleotide of claim 11 wherein A is biotin.
Claim 16
Dependent Claim
11 words
An oligo- or polynucleotide of claim 11 wherein A is iminobiotin.
Claim 17
Dependent Claim
17 words
An oligo- or polynucleotide of claims 14 or 15 wherein the linkage group is --CH═CH--CH 2 --NH--.
Claim 18
Dependent Claim
15 words
An oligo- or polynucleotide of claim 14 or 15 wherein the linkage group is ##STR29##
Claim 19
Dependent Claim
13 words
An oligo- or polynucleotide of claim 14 or 15 wherein B is uracil.
Claim 20
Dependent Claim
185 words
A method of making the oligo- or polynucleotide of claim 1 comprising the steps of: (a) reacting an oligo- or polynucleotide containing a nucleotide having the structure: ##STR30## with a mercuric salt in a suitable solvent under suitable conditions so as to form a mercurated derivative compound having the structure: ##STR31## (b) reacting said mercurated derivative 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 a palladium-containing compound under suitable conditions so as to form a compoundd having the structure: ##STR32## wherein N is a reactive terminal functional group or is A; and (c) when N is A, recovering said oligo- or polynucleotide of claim 1; 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 oligo- or polynucleotide of claim 1 and recovering said oligo- or polynucleotide.
Claim 21
Dependent Claim
47 words
A method in accordance with claim 19 wherein said suitable conditions comprise a pH in the range from about 4 to 8, a temperature in the range from about 20° C. to 30° C., and a reaction time in the range from about 3 to 24 hours.
Claim 22
Dependent Claim
14 words
A method in accordance with claim 19 wherein said mercuric salt is mercuric acetate.
Claim 23
Dependent Claim
22 words
A method in accordance with claim 19 wherein said chemical moiety represented by the formula . . . N is --CH═CH--CH.sub.2 --NH.sub.2.
Claim 24
Dependent Claim
21 words
A method in accordance with claim 19 wherein said chemical moiety represented by the formula . . . N is ##STR33##
Claim 25
Dependent Claim
14 words
A method in accordance with claim 19 wherein said aqueous solvents include a buffer.
Claim 26
Dependent Claim
31 words
A method in accordance with claim 24 wherein said buffer is selected from the group consisting of sodium acetate, potassium acetate, sodium citrate, potassium citrate, potassium citrate-phosphate, tris-acetate, and borate-sodium hydroxide.
Claim 27
Dependent Claim
19 words
A method in accordance with claim 25 wherein the concentration of said buffer is less than about 2.0 molar.
Claim 28
Dependent Claim
19 words
A method in accordance with claim 19 wherein in step (b) the aqueous solvent additionally includes an organic solvent.
Claim 29
Dependent Claim
13 words
A method in accordance with claim 27 wherein said organic solvent is water-miscible.
Claim 30
Dependent Claim
22 words
A method in accordance with claim 27 wherein said organic solvent is selected from the group consisting of alcohols, ketones, and amides.
Claim 31
Dependent Claim
27 words
A method in accordance with claim 29 wherein said organic solvent is selected from the group consisting of methanol, ethanol, propanol, glycerin, dioxane, acetone, pyridine, and dimethylformamide.
Claim 32
Dependent Claim
29 words
A method in accordance with claim 29 wherein A comprises biotin or iminibiotin, and M is selected from the group consisting of N-hydroxy-succinimide ester, imidate, anhydride, isothiocyanate, and epoxide.
Claim 33
Dependent Claim
13 words
A method in accordance with claim 19 wherein M is an N-hydroxysuccinimide ester.
Claim 34
Dependent Claim
21 words
A method in accordance with claim 19 wherein M is selected from the group consisting of imidate, anhydride, isothiocyanate, and epoxide.
Claim 35
Dependent Claim
25 words
A method in accordance with claim 19 wherein . . . N is selected from the group consisting of thiol, carboxylic acid, epoxide, and amine.
Claim 36
Dependent Claim
22 words
A method in accordance with claim 19 wherein said chemical moiety represented by the formula . . . N is --CH═CH--CH.sub.2 --NH-biotin.
Claim 37
Dependent Claim
21 words
A method in accordance with claim 19 wherein said chemical moiety represented by the formula . . . N is ##STR34##
Claim 38
Dependent Claim
22 words
A method in accordance with claim 19 wherein said chemical moiety represented by the formula . . . N is --CH═CH--CH.sub.2 --NH-iminobiotin.
Claim 39
Dependent Claim
21 words
A method in accordance with claim 19 wherein said chemical moiety represented by the formula . . . N is ##STR35##
Claim 40
Dependent Claim
13 words
A double-stranded polynucleotide duplex which includes the oligo- or polynucleotide of claim 1.
Claim 41
Dependent Claim
13 words
The duplex of claim 39 wherein said polynucleotide strands are ribonucleic acid molecules.
Claim 42
Dependent Claim
13 words
The duplex of claim 39 wherein said polynucleotide strands are deoxyribonucleic acid molecules.
Claim 43
Dependent Claim
142 words
An oligo- or polynucleotide sequence comprising at least one of a moiety having the structure: --BA wherein B represents a 7-deazapurine or a pyrimidine moiety suitable for incorporation into a polynucleotide and; wherein A comprises at least three carbon atoms and represents at least one component of a signalling moiety capable of producing a detectable signal; wherein B and A are covalently attached directly or through a linkage group that does not substantially interfere with the characteristic ability of the sequence to hybridize with a nucleic acid and does not substantially interfere with formation of the signalling moiety or detection of the detectable signal, provided also that if B is 7-deazapurine, A or the linkage group is attached to the 7-position of the deazapurine, and if B is pyrimidine, A or the linkage group is attached to the 5-position of the pyrimidine.
Claim 44
Dependent Claim
142 words
An oligo- or polynucleotide sequence comprising at least one of a moiety having the structure: --BA wherein B represents a 7-deazapurine or a pyrimidine moiety suitable for incorporation into a polynucleotide and; wherein A comprises at least three carbon atoms and represents at least one component of a signalling moiety capable of producing a detectable signal; wherein B and A are covalently attached directly or through a linkage group that does not substantially interfere with the characteristic ability of the sequence to hybridize with a nucleic acid and does not substantially interfere with formation of the signalling moiety or detection of the detectable signal, provided also that if B is 7-deazapurine, A or the linkage group is attached to the 7-position of the deazapurine, and if B is pyrimidine, A or the linkage group is attached to the 5-position of the pyrimidine.
Claim 45
Dependent Claim
13 words
An oligo- or polynucleotide sequence of claim 42 wherein A is a ligand.
Claim 46
Dependent Claim
13 words
An oligo- or polynucleotide sequence of claim 42 wherein A is a polypeptide.
Claim 47
Dependent Claim
27 words
An oligo- or polynucleotide sequence wherein A is fluorescent, electron dense, or is an enzyme capable of reacting with a substrate to form a detectable reaction product.
Claim 48
Dependent Claim
27 words
An oligo- or polynucleotide sequence wherein A is fluorescent, electron dense, or is an enzyme capable of reacting with a substrate to form a detectable reaction product.
Claim 49
Dependent Claim
20 words
An oligo- or polynucleotide sequence of claim 42 wherein A is a moiety consisting of at least five carbon atoms.
Claim 50
Dependent Claim
13 words
An oligo- or polynucleotide sequence of claim 42 wherein B is a pyrimidine.
Claim 51
Dependent Claim
185 words
An oligo- or polynucleotide containing therein a sugar moiety having the structure: ##STR36## wherein B represents a 7-deazapurine or a pyrimidine moiety covalently bonded to the C 1' -position of the sugar moiety, provided that whenever B is a 7-deazapurine, the sugar moiety is attached at the N 9 -position of the 7-deazapurine, and whenever B is a pyrimidin, the sugar moiety is attached at the N 1 -position of the pyrimidine; wherein A comprises at least three carbon atoms and represents at least one component of a signalling moiety capable of producing a detectable signal; and wherein B and A are covalently attached directly or through a linkage group that does not substantially interfere with the characteristic ability of the oligo- or polynucleotide to hybridize with a nucleic acid and does not substantially interfere with formation of the signalling moiety or detection of the detectable signal, provided also that if B is 7-deazapurine, A or the linkage group is attached to the 7-position of the deazapurine, and if B is pyrimidine, A or the linkage group is attached to the 5-position of the pyrimidine.
Claim 52
Dependent Claim
185 words
An oligo- or polynucleotide containing therein a sugar moiety having the structure: ##STR36## wherein B represents a 7-deazapurine or a pyrimidine moiety covalently bonded to the C 1' -position of the sugar moiety, provided that whenever B is a 7-deazapurine, the sugar moiety is attached at the N 9 -position of the 7-deazapurine, and whenever B is a pyrimidin, the sugar moiety is attached at the N 1 -position of the pyrimidine; wherein A comprises at least three carbon atoms and represents at least one component of a signalling moiety capable of producing a detectable signal; and wherein B and A are covalently attached directly or through a linkage group that does not substantially interfere with the characteristic ability of the oligo- or polynucleotide to hybridize with a nucleic acid and does not substantially interfere with formation of the signalling moiety or detection of the detectable signal, provided also that if B is 7-deazapurine, A or the linkage group is attached to the 7-position of the deazapurine, and if B is pyrimidine, A or the linkage group is attached to the 5-position of the pyrimidine.
Claim 53
Dependent Claim
27 words
An oligo- or polynucleotide of claim 48, wherein the sugar moiety has attached to its C 3' -position a member selected from the group consisting of: ##STR37##
Claim 54
Dependent Claim
27 words
An oligo- or polynucleotide of claim 48, wherein the sugar moiety has attached to its C 5' -position a member selected from the group consisting of: ##STR38##
Claim 55
Dependent Claim
23 words
An oligo- or polynucleotide of claim 48, wherein the sugar moiety has attached to its C 2' -position a H-- or HO-- group.
Claim 56
Dependent Claim
17 words
A method in accordance with claim 19, wherein the palladium-containing compound is K 2 PdCl 4 .
Claim 57
Dependent Claim
12 words
An oligo- or polynucleotide of claim 48 wherein A is a ligand.
Claim 58
Dependent Claim
28 words
An oligo- or polynucleotide of claim 3 or 53 wherein the ligand is selected from the group consisting of a hapten, an antigen, a cofactor, biotin and iminobiotin.
Claim 59
Dependent Claim
26 words
An oligo- or polynucleotide of claim 3 or 53 wherein the ligand is selected from the group consisting of dinitrophenol, lipoic acid and an olefinic compound.
Claim 60
Dependent Claim
24 words
An oligo- or polynucleotide of claim 3 or 53 wherein the ligand is capable of forming a complex by binding with a detectable polypeptide.
Claim 61
Dependent Claim
25 words
An oligo- or polynucleotide of claim 56 wherein the detectable polypeptide is selected from the group consisting of an antibody, an enzyme, streptavidin and avidin.
Claim 62
Dependent Claim
20 words
An oligo- or polynucleotide of claim 56 wherein an indicator molecule is associated with or bound to the detectable polypeptide.
Claim 63
Dependent Claim
24 words
An oligo- or polynucleotide of claim 58 wherein the indicator molecule is fluorescent, electron dense, or an enzyme capable of depositing insoluble reaction products.
Claim 64
Dependent Claim
22 words
An oligo- or polynucleotide of claim 59 wherein the enzyme is selected from the group consisting of alkaline phosphatase, peroxidase and β-galactosidase.
Claim 65
Dependent Claim
22 words
An oligo- or polynucleotide of claim 59 wherein the fluorescent indicator molecule is selected from the group consisting of fluorescein and rhodamine.
Claim 66
Dependent Claim
25 words
An oligo- or polynucleotide of claim 59 wherein the electron dense indicator molecule is selected from the group consisting of ferritin, hemocyanin and colloidal gold.
Claim 67
Dependent Claim
18 words
An oligo- or polynucleotide of claim 58 wherein the indicator molecule is covalently linked to the detectable polypeptide.
Claim 68
Dependent Claim
30 words
An oligo- or polynucleotide of claim 58 wherein the detectable polypeptide is indirectly detectable by specifically complexing the detectable polypeptide with a second polypeptide covalently linked to an indicator molecule.
Claim 69
Dependent Claim
35 words
An oligo- or polynucleotide of claim 64 wherein said detectable polypeptide is selected form the group consisting of avidin and streptavidin and the second polypeptide is selected from the group consisting of biotin and iminobiotin.
Claim 70
Dependent Claim
32 words
An oligo- or polynucleotide of claim 56 wherein at least one compound is labeled with a first indicator molecule and at least one other compound is labeled with a second indicator molecule.
Claim 71
Dependent Claim
15 words
An oligo- or polynucleotide of claim 1 or 48 wherein A comprises an indicator molecule.
Claim 72
Dependent Claim
25 words
An oligo- or polynucleotide of claim 67 wherein said indicator molecule is fluorescent, electron dense, or is an enzyme capable of depositing insoluble reaction products.
Claim 73
Dependent Claim
22 words
An oligo- or polynucleotide of claim 68 wherein the enzyme is selected from the group consisting of alkaline phosphatase, peroxidase and β-galactosidase.
Claim 74
Dependent Claim
22 words
An oligo- or polynucleotide of claim 68 wherein the fluorescent indicator molecule is selected from the group consisting of fluorescein and rhodamine.
Claim 75
Dependent Claim
24 words
An oligo- or polynucleotide of claim 68 wherein the electron dense compound is selected from the group consisting of ferritin, hemocyanin and colloidal gold.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1998-09-11 | 1999-03-13 | 1999-09-12 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2002-09-11 | 2003-03-13 | 2003-09-12 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2006-09-11 | 2007-03-13 | 2007-09-12 | $7700.00 | Window Closed / Lapsed |