Process for labeling nucleic acids using psoralen derivatives

• US 4,582,789 A
• Filed: 12/18/1984
• Issued: 04/15/1986
• Est. Priority Date: 03/21/1984
• Status: Expired due to Term

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Cetus Corporation (Novartis Ag)
• Inventors: Sheldon, Edward L. III, Rapoport, Henry, Levenson, Corey H., Mullis, Kary B.
• Primary Examiner(s): Nucker, Christine M.
• Application Number:
  US06/683,263
• Time in Patent Office: 483 Days
• Field of Search: 435/6, 435/7, 436/501, 935/77, 935/78
• US Class Current: 435/6.18
• CPC Class Codes: C07D 493/04 Ortho-condensed systems
• CPC Class Codes: C07D 513/04 Ortho-condensed systems
• 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

A labeling reagent of the formula:

[A][B]L

is prepared where A is an alkylating intercalation moiety, B is a divalent organic spacer arm moiety with a straight chain of at least two carbon atoms, and L is a monovalent label moiety capable of producing a detectable signal, e.g., a signal detectable by spectroscopic, photochemical, chemical, immunochemical or biochemical means. Preferably A is a 4'"'"'-methylene-substituted psoralen moiety, and most preferably A is a 4'"'"'-methylene-substituted 4,5'"'"',8-trimethylpsoralen moiety.

This reagent may be used to label nucleic acids, preferably DNA, by intercalating the alkylating intercalation moiety of the reagent into an at least partially double-stranded nucleic acid to form a complex and activating the complex to cause covalent bonding between the reagent and the nucleic acid. Preferably, the labeled nucleic acid is a hybridization probe for detecting nucleic acid sequences capable of hybridizing with a hydridizing region of the nucleic acid. Also preferably the label moiety is non-radioactive.

This reagent may also be used in chromosome banding to label specific regions of chromosomes and thereby differentiate them.

Claims

1. A composition for labeling nucleic acids of the formula:

space="preserve" listing-type="equation">[A--[B--L

wherein A is an alkylating intercalation moiety, B is a divalent organic moiety having the formula;

##STR21## where R is --H or a --CHO group, R" is --H, x is a number from 1 to 4, and y is a number from 2 to 4, and L is a monovalent label moiety, and wherein B is exclusive of any portion of the intercalation and label moieties.

2. The composition of claim 1 wherein A is a 4'"'"'-methylene-substituted psoralen moiety.

3. The composition of claim 2 wherein A is of the formula:

##STR22##

4. The composition of claim 1 wherein R is --CHO, x is 2 and y is 2.

5. The composition of claim 1 wherein L is detectable by spectroscopy, radioactivity, photochemistry, or by biochemical, immunochemical or chemical means.

6. The composition of claim 5 wherein L is detectable by fluorescent or chromogenic detection means.

7. The composition of claim 5 wherein L is detectable by formation of a complex between L and a polypeptide, lectin or antibody with or without an enzyme associated therewith.

8. The composition of claim 1 having the formula:

##STR23## where R and R'"'"' are independently --CH₃ or --H.

9. The composition of claim 1 having the formula:

##STR24## where R and R'"'"' are independently --CH₃ or --H.

10. The composition of claim 1 having the formula:

##STR25##

11. The composition of claim 1 having the formula:

##STR26##

12. The composition of claim 1 having the formula:

##STR27##

13. In a process for labeling specific regions of chromosomes wherein the chromosome regions are stained differentially with a detectable reagent so as to distinguish the chromosomes and the reagent is detected, the improvement which comprises employing as the reagent the composition of claim 6.