Linkage of proteins to nucleic acids

• US 4,587,044 A
• Filed: 09/01/1983
• Issued: 05/06/1986
• Est. Priority Date: 09/01/1983
• Status: Expired due to Fees

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Ts'O, Paul O. P., Miller, Paul S.
• Primary Examiner(s): Kight, John
• Assistant Examiner(s): Draper, Garnette D.
• Application Number:
  US06/528,573
• Time in Patent Office: 978 Days
• Field of Search: 260/112 R, 260/112.5 R, 260/112 B, 536/22, 536/28, 536/29, 424/85, 435/6, 435/177, 435/180, 435/181
• US Class Current: 530/322
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 14/42 Lectins, e.g. concanavalin, phytohaemagglutinin
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence

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.

Claims

1. 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 H₂ N--(CH₂)_n --NH₂ 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.

2. The method of claim 1 wherein said bifunctional crosslinking agent is a homobifunctional acylating agent.

3. The method of claim 2 wherein said homobifunctional acylating agent is dithiobis (N-hydroxysuccinimidylpropionate).

4. The method of claim 1 wherein said bifunctional crosslinking agent is a heterobifunctional acylating agent.

5. The method of claim 4 wherein said heterobifunctional acylating agent is N-succinimidyl 2-pyridyldithiopropionate.

6. The method of claim 4 wherein said heterobifunctional acylating agent is N-succinimidyl-4'"'"'-(p-maleimidophenyl) butyrate.

7. 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.

8. The method of claim 7 wherein the biotin-containing acylating agent is biotinyl N-hydroxysuccinimide.

9. 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.

47. A nucleic acid-protein conjugate made in accordance with any one of claims 1-46.

10. 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.

11. The method of claim 10 wherein said bifunctional crosslinking agent is a homobifunctional acylating agent.

12. The method of claim 11 wherein said homobifunctional acylating agent is dithiobis(N-hydroxysuccinimidylpropionate).

13. The method of claim 10 wherein said bifunctional crosslinking agent is a heterobifunctional acylating agent.

14. The method of claim 13 wherein said heterobifunctional acylating agent is N-succinimidyl 2-pyridyldithiopropionate.

15. The method of claim 13 wherein said heterobifunctional acylating agent is N-succinimidyl-4'"'"'-(p-maleimidophenyl) butyrate.

16. 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.

17. 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.

18. The method of claim 17 wherein said bifunctional crosslinking agent is a homobifunctional acylating agent.

19. The method of claim 18 wherein said homobifunctional acylating agent is dithiobis (N-hydroxysuccinimidylpropionate).

20. The method of claim 17 wherein said bifunctional crosslinking agent is a heterobifunctional acylating agent.

21. The method of claim 20 wherein said heterobifunctional acylating agent is N-succinimidyl 2-pyridyldithiopropionate.

22. The method of claim 20 wherein said heterobifunctional acylating agent is N-succinimidyl-4'"'"'-(p-maleimidophenyl) butyrate.

23. 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.

24. The method of claim 23 wherein said bifunctional crosslinking agent is a homobifunctional acylating agent.

25. The method of claim 24 wherein said homobifunctional acylating agent is dithiobis (N-hydroxysuccinimidylpropionate).

26. The method of claim 23 wherein said bifunctional crosslinking agent is a heterobifunctional acylating agent.

27. The method of claim 26 wherein said heterobifunctional acylating agent is N-succinimidyl 2-pyridyldithiopropionate.

28. The method of claim 26 wherein said heterobifunctional acylating agent is N-succinimidyl-4'"'"'-(p-maleimidophenyl) butyrate.

29. 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.

30. The method of claim 29 wherein said bifunctional crosslinking agent is a homobifunctional acylating agent.

31. The method of claim 30 wherein said homobifunctional acylating agent is dithiobis (N-hydroxysuccinimidylpropionate).

32. The method of claim 29 wherein said bifunctional crosslinking agent is a heterobifunctional acylating agent.

33. The method of claim 32 wherein said heterobifunctional acylating agent is N-succinimidyl 2-pyridyldithiopropionate.

34. The method of claim 32 wherein said heterobifunctional acylating agent is N-succinimidyl-4'"'"'-(p-maleimidophenyl) butyrate.

35. 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.

36. The method of claim 35 wherein said bifunctional crosslinking agent is a homobifunctional acylating agent.

37. The method of claim 36 wherein said homobifunctional acylating agent is dithiobis (N-hydroxysuccinimidylpropionate).

38. The method of claim 35 wherein said bifunctional crosslinking agent is a heterobifunctional acylating agent.

39. The method of claim 38 wherein said heterobifunctional acylating agent is N-succinimidyl 2-pyridyldithiopropionate.

40. The method of claim 38 wherein said heterobifunctional acylating agent is N-succinimidyl-4'"'"'-(p-maleimidophenyl) butyrate.

41. 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.

42. The method of claim 41 wherein said bifunctional crosslinking agent is a homobifunctional acylating agent.

43. The method of claim 42 wherein said homobifunctional acylating agent is dithiobis(N-hydroxysuccinimidylpropionate).

44. The method of claim 41 wherein said bifunctional crosslinking agent is a heterobifunctional acylating agent.

45. The method of claim 44 wherein said heterobifunctional acylating agent is N-succinimidyl 2-pyridyldithiopropionate.

46. The method of claim 44 wherein said heterobifunctional acylating agent is N-succinimidyl-4'"'"'-(p-maleimidophenyl) butyrate.