Pro-thiol aryl azide labelling of nucleic acids

• US 5,854,409 A
• Filed: 04/08/1997
• Issued: 12/29/1998
• Est. Priority Date: 11/27/1995
• Status: Expired due to Term

• Current Assignee: Vector Laboratories
• Original Assignee: Vector Laboratories
• Inventors: Westling, Mark E., Daniel, Steven G.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/835,267
• Time in Patent Office: 630 Days
• Field of Search: 536/22.1, 536/23.1, 536/24.1, 536/24.3-33, 536/25.3, 552/1, 552/8, 564/1, 564/305, 564/440
• US Class Current: 536/22.1
• 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

Abstract

Reagents and methods for multi-step labeling of nucleic acids allow the addition of relatively insoluble or unstable labels to nucleic acid in the final step. Nucleic acids can be stored as a stable intermediate capable of reacting with a label conjugated to a thiol-reactive group.

Claims

1. A pro-thiol aryl azide for coupling a thiol group to a nucleic acid, said pro-thiol aryl azide having the structure:

##STR40## wherein, R₁ to R₄, which may be the same or different, are selected from the group consisting of H, alkyl, nitro, hydroxy, halide, carboxylic acid, methoxy, cyano, amino groups and salts thereof;

wherein E comprises an amine and salts thereof, and a chain of C₁ -C₂₀ having chain constituents selected from the group consisting of alkyl, alkenyl, alkynyl heteroatoms and salts thereof, said chain constituents having substituents selected from the group consisting of H, C₁ -C₆ alkyl, aryl, ester, ether, amine, amide, halo groups and salts thereof;

and wherein Y is a group capable of protecting the sulfur moiety from chemical reaction and removable so as to generate a thiol moiety.

2. The pro-thiol aryl azide according to claim 1 wherein said heteroatoms are selected from the group consisting of N, O and S.

3. The pro-thiol aryl azide according to claim 1 wherein R₁, R₂ and R₄ are hydrogen and R₃ is nitro.

4. The pro-thiol aryl azide according to claim 1 wherein Y is selected from the group consisting of:

##STR41## wherein R_a, R_b, R_c, R_d, R_e and R_f, which may be the same or different, are selected from the group consisting of hydrogen, alkyl of 1 to 6 carbons or alkoxy of 1 to 6 carbons;

wherein R_g, R_h and R_i, which may be the same or different, are selected from the group consisting of alkyl of 1 to 6 carbons and aryl; and

wherein R_j is selected from the group consisting of alkyl, aryl, alkyl containing heteroatoms or aryl containing heteroatoms.

5. The pro-thiol aryl azide according to claim 3 wherein E is:

##STR42## and Y is;

##STR43##

6. A covalent conjugate of a nucleic acid and a thiol-reactive label having a thiol-reactive group given by the structure:

##STR44## wherein, R₁ to R₄, which may be the same or different, are selected from the group consisting of H, alkyl, nitro, hydroxy, halide, carboxylic acid, methoxy, cyano, amino groups and salts thereof;

wherein E comprises an amine and salts thereof, and a chain of C₁ -C₂₀ having chain constituents selected from the group consisting of alkyl, alkenyl, alkynyl heteroatoms and salts thereof, said chain constituents having substituents selected from the group consisting of H, C₁ -C₆ alkyl, aryl, ester, ether, amine, amide, halo groups and salts thereof;

wherein R₉ is the thiolated conjugate of the thiol-reactive group in the thio-reactive label; and

wherein L is selected from the group consisting of a ligand or a detectable label.

7. The conjugate of claim 6, R₁, R₂ and R₄ are hydrogen and R₃ is nitro.

8. The conjugate of claim 7 wherein E is represented by the structure:

##STR45##

9. The conjugate of claim 6, wherein the thiol reactive group is selected from the group consisting of maleimides, iodoacetamides, pyridyl disulfides, vinyl pyridines, vinyl sulfones, acrylates, aryl mercurial compounds and aziridino compounds covalently linked to L.

10. The conjugate of claim 6, wherein L is selected from the group consisting of fluorophores, biotin, fucose and haptens.

11. The conjugate of claim 6, wherein the thiol reactive group is a maleimide and L is fluorescein.

12. The conjugate of claim 6, wherein said nucleic acid is selected from the group consisting of native sequences, synthetic nucleic acids, synthetic nucleic acid analogs, or sequences derived by cloning.

13. The conjugate of claim 12, wherein said synthetic nucleic acid analog is selected from the group consisting of peptide nucleic acid or phosphorothioates.

14. A pro-thiol aryl azide for coupling a thiol group to a nucleic acid, said pro-thiol aryl azide having the structure:

##STR46## wherein, R₁ to R₄, which may be the same or different, are selected from the group consisting of H, alkyl, nitro, hydroxy, halide, carboxylic acid, methoxy, cyano, amino groups and salts thereof;

wherein E is;

##STR47## and Y is;

##STR48##

15. A covalent conjugate of a nucleic acid and a thiol-reactive label having a thiol-reactive group given by the structure:

##STR49## wherein, R₁ to R₄, which may be the same or different, are selected from the group consisting of H, alkyl, nitro, hydroxy, halide, carboxylic acid, methoxy, cyano, amino groups and salts thereof;

wherein E is;

##STR50## wherein R₉ is the thiolated conjugate of the thiol-reactive group in the thio-reactive label; and

wherein L is selected from the group consisting of a ligand or a detectable label.