Patent US4587044A successfully refreshed from Google Patents.
US4587044A
Expired / Lapsed
US
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Linkage of proteins to nucleic acids
Assignee: PARAMOUNT CAPITAL Inc
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Filing: 1983-09-01
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Granted: 1986-05-06
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Expires: 2003-09-01
Abstract
Abstract A nucleic acid-protein conjugate which is specific with respect to a selected living cell is prepared by linking said nucleic acid to a protein specific to said living cell.
Inventors
- 👤 Paul S. Miller
- 👤 Paul O. P. Ts'o
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 14:07
CPC Classifications
C; C07; C07H; C07H21/00; C07H19/00; C07H19/02; C07H19/04; C07H19/06; C07H19/10; C07H19/16; C07H19/20; C07K; C07K1/00; C07K1/107; C07K1/1072; C07K1/1077; C07K14/00; C07K14/415; C07K14/42; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; A; A61; A61K; A61K38/00
Claims Analysis
Total Claims: 54
Claim 1
Independent Claim
126 words
A method for producing a nucleic acid-protein conjugate which interacts with a living cell wherein the protein is specifically selected for the purpose of guiding the nucleic acid into said living cell, said method comprising (a) modifying said nucleic acid by esterifying the said nucleic acid with a saturated or unsaturated aliphatic dicarboxylic acid or an anhydride thereof and reacting the terminal free carboxyl group of the resulting ester with a diaminoalkane of the formula H 2 N--(CH 2 ) n --NH 2 wherein n is an integer from 2 to 12, so as to produce a modified nucleic acid containing a terminal amino group reactive with a bifunctional crosslinking agent and (b) reacting said bifunctional crosslinking agent with said modified nucleic acid and said protein.
Claim 2
Independent Claim
126 words
A method for producing a nucleic acid-protein conjugate which interacts with a living cell wherein the protein is specifically selected for the purpose of guiding the nucleic acid into said living cell, said method comprising (a) modifying said nucleic acid by esterifying the said nucleic acid with a saturated or unsaturated aliphatic dicarboxylic acid or an anhydride thereof and reacting the terminal free carboxyl group of the resulting ester with a diaminoalkane of the formula H 2 N--(CH 2 ) n --NH 2 wherein n is an integer from 2 to 12, so as to produce a modified nucleic acid containing a terminal amino group reactive with a bifunctional crosslinking agent and (b) reacting said bifunctional crosslinking agent with said modified nucleic acid and said protein.
Claim 3
Dependent Claim
15 words
The method of claim 1 wherein said bifunctional crosslinking agent is a homobifunctional acylating agent.
Claim 4
Dependent Claim
13 words
The method of claim 2 wherein said homobifunctional acylating agent is dithiobis (N-hydroxysuccinimidylpropionate).
Claim 5
Dependent Claim
15 words
The method of claim 1 wherein said bifunctional crosslinking agent is a heterobifunctional acylating agent.
Claim 6
Dependent Claim
13 words
The method of claim 4 wherein said heterobifunctional acylating agent is N-succinimidyl 2-pyridyldithiopropionate.
Claim 7
Dependent Claim
13 words
The method of claim 4 wherein said heterobifunctional acylating agent is N-succinimidyl-4'-(p-maleimidophenyl) butyrate.
Claim 8
Dependent Claim
44 words
The method of claim 1 wherein the amino group of said modified nucleic acid and the amino groups of said protein are reacted with a biotin-containing acylating agent and thereafter the biotin moieties of each are reacted with glycoprotein avidin or vitamin H avidin.
Claim 9
Dependent Claim
13 words
The method of claim 7 wherein the biotin-containing acylating agent is biotinyl N-hydroxysuccinimide.
Claim 10
Dependent Claim
41 words
The method of claim 1 wherein the amino group of said modified nucleic acid and the amino groups of said protein are reacted with a biotin-containing acylating agent and thereafter the biotin moieties of each are reacted with a monoclonal antibody.
Claim 11
Independent Claim
104 words
A method for producing a nucleic acid-protein conjugate which interacts with a living cell wherein the protein is specifically selected for the purpose of guiding the nucleic acid into said living cell, said method comprising (a) modifying said nucleic acid by esterifying said nucleic acid with a saturated or unsaturated aliphatic dicarboxylic acid or an anhydride thereof and reacting the terminal free carboxyl group of the resulting ester with cysteamine so as to produce a modified nucleic acid containing a terminal thio group reactive with a bifunctional crosslinking agent, and (b) reacting said bifunctional crosslinking agent with said modified nucleic acid and said protein.
Claim 12
Independent Claim
104 words
A method for producing a nucleic acid-protein conjugate which interacts with a living cell wherein the protein is specifically selected for the purpose of guiding the nucleic acid into said living cell, said method comprising (a) modifying said nucleic acid by esterifying said nucleic acid with a saturated or unsaturated aliphatic dicarboxylic acid or an anhydride thereof and reacting the terminal free carboxyl group of the resulting ester with cysteamine so as to produce a modified nucleic acid containing a terminal thio group reactive with a bifunctional crosslinking agent, and (b) reacting said bifunctional crosslinking agent with said modified nucleic acid and said protein.
Claim 13
Dependent Claim
15 words
The method of claim 10 wherein said bifunctional crosslinking agent is a homobifunctional acylating agent.
Claim 14
Dependent Claim
12 words
The method of claim 11 wherein said homobifunctional acylating agent is dithiobis(N-hydroxysuccinimidylpropionate).
Claim 15
Dependent Claim
15 words
The method of claim 10 wherein said bifunctional crosslinking agent is a heterobifunctional acylating agent.
Claim 16
Dependent Claim
13 words
The method of claim 13 wherein said heterobifunctional acylating agent is N-succinimidyl 2-pyridyldithiopropionate.
Claim 17
Dependent Claim
13 words
The method of claim 13 wherein said heterobifunctional acylating agent is N-succinimidyl-4'-(p-maleimidophenyl) butyrate.
Claim 18
Dependent Claim
36 words
The method of claim 10 wherein the protein is acylated with, as a heterobifunctional reagent, N-succinimidyl-4'-(p-maleimidophenyl) butyrate and the terminal thio group of said modified nucleic acid reacts with the maleimido moiety of said acylated protein.
Claim 19
Independent Claim
108 words
A method for producing a nucleic acid-protein conjugate which interacts with a living cell wherein the protein is specifically selected for the purpose of guiding the nucleic acid into said living cell, said method comprising (a) modifying said nucleic acid by esterifying said nucleic acid with a saturated or unsaturated aliphatic dicarboxylic acid or an anhydride thereof and reacting the terminal free carboxyl group of the resulting ester with 2,2'-dithiobis (aminoethane) so as to produce a modified nucleic acid having a terminal disulfide group adaptable to be reactive with a bifunctional crosslinking agent, and (b) reacting said bifunctional crosslinking agent with said modified nucleic acid and said protein.
Claim 20
Independent Claim
108 words
A method for producing a nucleic acid-protein conjugate which interacts with a living cell wherein the protein is specifically selected for the purpose of guiding the nucleic acid into said living cell, said method comprising (a) modifying said nucleic acid by esterifying said nucleic acid with a saturated or unsaturated aliphatic dicarboxylic acid or an anhydride thereof and reacting the terminal free carboxyl group of the resulting ester with 2,2'-dithiobis (aminoethane) so as to produce a modified nucleic acid having a terminal disulfide group adaptable to be reactive with a bifunctional crosslinking agent, and (b) reacting said bifunctional crosslinking agent with said modified nucleic acid and said protein.
Claim 21
Dependent Claim
15 words
The method of claim 17 wherein said bifunctional crosslinking agent is a homobifunctional acylating agent.
Claim 22
Dependent Claim
13 words
The method of claim 18 wherein said homobifunctional acylating agent is dithiobis (N-hydroxysuccinimidylpropionate).
Claim 23
Dependent Claim
15 words
The method of claim 17 wherein said bifunctional crosslinking agent is a heterobifunctional acylating agent.
Claim 24
Dependent Claim
13 words
The method of claim 20 wherein said heterobifunctional acylating agent is N-succinimidyl 2-pyridyldithiopropionate.
Claim 25
Dependent Claim
13 words
The method of claim 20 wherein said heterobifunctional acylating agent is N-succinimidyl-4'-(p-maleimidophenyl) butyrate.
Claim 26
Independent Claim
118 words
A method for producing a nucleic acid-protein conjugate which interacts with a living cell wherein the protein is specifically selected for the purpose of guiding the nucleic acid into said living cell, said nucleic acid containing cytosine, said method comprising (a) modifying said cytosine containing nucleic acid by transaminating said nucleic acid by reacting it with a diaminoalkane in the presence of an alkali metal bisulfite whereby the bisulfite forms an adduct with said cytosine and whereby the diaminoalkane displaces the C 4-amino group so as to produce a modified nucleic acid having a terminal aminoalkyl group reactive with a bifunctional crosslinking agent, and (b) reacting said bifunctional crosslinking agent with said modified nucleic acid and said protein.
Claim 27
Independent Claim
118 words
A method for producing a nucleic acid-protein conjugate which interacts with a living cell wherein the protein is specifically selected for the purpose of guiding the nucleic acid into said living cell, said nucleic acid containing cytosine, said method comprising (a) modifying said cytosine containing nucleic acid by transaminating said nucleic acid by reacting it with a diaminoalkane in the presence of an alkali metal bisulfite whereby the bisulfite forms an adduct with said cytosine and whereby the diaminoalkane displaces the C 4-amino group so as to produce a modified nucleic acid having a terminal aminoalkyl group reactive with a bifunctional crosslinking agent, and (b) reacting said bifunctional crosslinking agent with said modified nucleic acid and said protein.
Claim 28
Dependent Claim
15 words
The method of claim 23 wherein said bifunctional crosslinking agent is a homobifunctional acylating agent.
Claim 29
Dependent Claim
13 words
The method of claim 24 wherein said homobifunctional acylating agent is dithiobis (N-hydroxysuccinimidylpropionate).
Claim 30
Dependent Claim
15 words
The method of claim 23 wherein said bifunctional crosslinking agent is a heterobifunctional acylating agent.
Claim 31
Dependent Claim
13 words
The method of claim 26 wherein said heterobifunctional acylating agent is N-succinimidyl 2-pyridyldithiopropionate.
Claim 32
Dependent Claim
13 words
The method of claim 26 wherein said heterobifunctional acylating agent is N-succinimidyl-4'-(p-maleimidophenyl) butyrate.
Claim 33
Independent Claim
121 words
A method for producing a nucleic acid-protein conjugate which interacts with a living cell wherein the protein is specifically selected for the purpose of guiding the nucleic acid into said living cell, said nucleic acid containing cytosine, said method comprising (a) modifying said cytosine containing nucleic acid by transaminating said nucleic acid by reacting it with cysteamine in the presence of an alkali metal bisulfite whereby the bisulfite forms an adduct with said cytosine and whereby the amino group of the cysteamine displaces the C 4-amino group so as to produce a modified nucleic acid having a terminal thiol group reactive with a bifunctional crosslinking agent, and (b) reacting said bifunctional crosslinking agent with said modified nucleic acid and said protein.
Claim 34
Independent Claim
121 words
A method for producing a nucleic acid-protein conjugate which interacts with a living cell wherein the protein is specifically selected for the purpose of guiding the nucleic acid into said living cell, said nucleic acid containing cytosine, said method comprising (a) modifying said cytosine containing nucleic acid by transaminating said nucleic acid by reacting it with cysteamine in the presence of an alkali metal bisulfite whereby the bisulfite forms an adduct with said cytosine and whereby the amino group of the cysteamine displaces the C 4-amino group so as to produce a modified nucleic acid having a terminal thiol group reactive with a bifunctional crosslinking agent, and (b) reacting said bifunctional crosslinking agent with said modified nucleic acid and said protein.
Claim 35
Dependent Claim
15 words
The method of claim 29 wherein said bifunctional crosslinking agent is a homobifunctional acylating agent.
Claim 36
Dependent Claim
13 words
The method of claim 30 wherein said homobifunctional acylating agent is dithiobis (N-hydroxysuccinimidylpropionate).
Claim 37
Dependent Claim
15 words
The method of claim 29 wherein said bifunctional crosslinking agent is a heterobifunctional acylating agent.
Claim 38
Dependent Claim
13 words
The method of claim 32 wherein said heterobifunctional acylating agent is N-succinimidyl 2-pyridyldithiopropionate.
Claim 39
Dependent Claim
13 words
The method of claim 32 wherein said heterobifunctional acylating agent is N-succinimidyl-4'-(p-maleimidophenyl) butyrate.
Claim 40
Independent Claim
125 words
A method for producing a nucleic acid-protein conjugate which interacts with a living cell wherein the protein is specifically selected for the purpose of guiding the nucleic acid into said living cell, said nucleic acid containing cytosine, said method comprising (a) modifying said cytosine containing nucleic acid by transaminating said nucleic acid by reacting it with 2,2'-dithiobis(aminoethane) in the presence of an alkali metal bisulfite whereby the bisulfite forms an adduct with said cytosine and whereby the amino group of said 2,2'-dithiobis (aminoethane) displaces the C 4-amino group so as to produce a modified nucleic acid having a terminal disulfide group adaptable to be reactive with a bifunctional crosslinking agent, and (b) reacting said bifunctional crosslinking agent with said modified nucleic acid and said protein.
Claim 41
Independent Claim
125 words
A method for producing a nucleic acid-protein conjugate which interacts with a living cell wherein the protein is specifically selected for the purpose of guiding the nucleic acid into said living cell, said nucleic acid containing cytosine, said method comprising (a) modifying said cytosine containing nucleic acid by transaminating said nucleic acid by reacting it with 2,2'-dithiobis(aminoethane) in the presence of an alkali metal bisulfite whereby the bisulfite forms an adduct with said cytosine and whereby the amino group of said 2,2'-dithiobis (aminoethane) displaces the C 4-amino group so as to produce a modified nucleic acid having a terminal disulfide group adaptable to be reactive with a bifunctional crosslinking agent, and (b) reacting said bifunctional crosslinking agent with said modified nucleic acid and said protein.
Claim 42
Dependent Claim
15 words
The method of claim 35 wherein said bifunctional crosslinking agent is a homobifunctional acylating agent.
Claim 43
Dependent Claim
13 words
The method of claim 36 wherein said homobifunctional acylating agent is dithiobis (N-hydroxysuccinimidylpropionate).
Claim 44
Dependent Claim
15 words
The method of claim 35 wherein said bifunctional crosslinking agent is a heterobifunctional acylating agent.
Claim 45
Dependent Claim
13 words
The method of claim 38 wherein said heterobifunctional acylating agent is N-succinimidyl 2-pyridyldithiopropionate.
Claim 46
Dependent Claim
13 words
The method of claim 38 wherein said heterobifunctional acylating agent is N-succinimidyl-4'-(p-maleimidophenyl) butyrate.
Claim 47
Independent Claim
63 words
A method for producing a nucleic acid-protein conjugate which interacts with a living cell wherein the protein is specifically selected for the purpose of guiding the nucleic acid into said living cell, said method comprising (a) modifying said nucleic acid enzymatically to incorporate aminoethyldeoxycytidine or aminoethylcytidine residues therein and (b) reacting a bifunctional crosslinking agent with said modified nucleic acid and said protein.
Claim 48
Independent Claim
63 words
A method for producing a nucleic acid-protein conjugate which interacts with a living cell wherein the protein is specifically selected for the purpose of guiding the nucleic acid into said living cell, said method comprising (a) modifying said nucleic acid enzymatically to incorporate aminoethyldeoxycytidine or aminoethylcytidine residues therein and (b) reacting a bifunctional crosslinking agent with said modified nucleic acid and said protein.
Claim 49
Dependent Claim
15 words
The method of claim 41 wherein said bifunctional crosslinking agent is a homobifunctional acylating agent.
Claim 50
Dependent Claim
12 words
The method of claim 42 wherein said homobifunctional acylating agent is dithiobis(N-hydroxysuccinimidylpropionate).
Claim 51
Dependent Claim
15 words
The method of claim 41 wherein said bifunctional crosslinking agent is a heterobifunctional acylating agent.
Claim 52
Dependent Claim
13 words
The method of claim 44 wherein said heterobifunctional acylating agent is N-succinimidyl 2-pyridyldithiopropionate.
Claim 53
Dependent Claim
13 words
The method of claim 44 wherein said heterobifunctional acylating agent is N-succinimidyl-4'-(p-maleimidophenyl) butyrate.
Claim 54
Dependent Claim
13 words
A nucleic acid-protein conjugate made in accordance with any one of claims 1-46.
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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) | 1989-05-05 | 1989-11-04 | 1990-05-06 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-05-05 | 1993-11-04 | 1994-05-06 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-05-05 | 1997-11-04 | 1998-05-06 | $7700.00 | Window Closed / Lapsed |