Multichromophore fluorescent probes using DNA intercalation complexes

• US 6,280,933 B1
• Filed: 11/07/1997
• Issued: 08/28/2001
• Est. Priority Date: 03/14/1990
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Mathies, Richard A., Glazer, Alexander N., Peck, Konan
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/966,398
• Time in Patent Office: 1,390 Days
• Field of Search: 435/6, 435/91.2, 536/26.6, 536/23.1
• US Class Current: 435/6.14
• CPC Class Codes: C07D 221/12 Phenanthridines
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 417/06 linked by a carbon chain containing only aliphatic carbon atoms
• CPC Class Codes: C07D 417/14 containing three or more hetero rings
• CPC Class Codes: C09B 23/04 one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules
• CPC Class Codes: G01N 27/44747 Composition of gel or of carrier mixture
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Novel fluorescent labeling techniques and fluorescent labels are provided, employing high affinity non-covalently binding and intercalating fluorescent dyes and dsDNA. The dyes find application to provide highly sensitive labeling of nucleic acids in electrophoretic gels and as pre-prepared labels for binding to a wide variety of specific binding pair members. The DNA-dye fluorescer complex can be used for labels in diagnostic assays, detection of specific nucleic acid sequences, and the like.

Claims

1. A fluorescent label comprising (a) a nucleic acid of at least about 20 nucleotides and (b) non-covalently bound and intercalated in said nucleic acid at least one fluorescent molecule characterized by having at least two positive charges, having at least one fluorescent monomeric unit and having a binding affinity to double stranded DNA of at least about 5×

10⁶ M<sup>−

1</sup> and up to about 1 fluorescent unit per 4 nucleotides in a nucleic acid strand,

wherein said fluorescent unit is characterized by having at least one aromatic ring per monomeric unit, and

wherein said nucleic acid comprises a member of a specific binding pair capable of binding to a complementary member of said specific binding pair or a functionality for covalent bonding to another molecule other than a naturally occurring functionality of a nucleotide.

2. A fluorescent label according to claim 1, wherein said fluorescent molecule is a multimer.

3. A fluorescent molecule according to claim 2, wherein said multimer is a dimer and at least one of said monomeric units is a phenanthridine.

4. A fluorescent label according to claim 3, wherein said dimer is an ethidium diner.

5. A fluorescent label according to claim 2, wherein said multimer is a dimer and at least one of said monomeric units is an acridine.

6. A fluorescent label according to claim 1, wherein said nucleic acid is double stranded DNA and said ratio of fluorescent molecule to double stranded DNA is in the range of 4-5 base pairs per fluorescent molecule.

14. A complex of a fluorescent label according to claim 1 comprising a member of a specific binding pair and its complementary binding pair member.

15. A complex according to claim 14, wherein said complementary binding pair member is a ligand.

16. A complex according to claim 14, wherein said complementary binding pair member is a naturally occurring receptor or an antibody.

17. A complex according to claim 14, wherein said complementary binding pair member is a nucleic acid strand.

19. The composition according to any one of claim 1, 7 or 18, wherein said nucleic acid in said label composition further comprises single stranded nucleic acid.

7. A fluorescent label comprising:

(a) double stranded DNA,

(b) a fluorescent aromatic dimer having two monomeric fluorescent units joined by a chain of at least about 10 Å

non-covalently bound and intercalated in said double stranded DNA at a ratio of at least about 1;

5 base pairs, and

(c) covalently joined to said double stranded DNA a ligand member of a specific binding pair.

8. A fluorescent label according to claim 7, wherein said ligand member is biotin.

9. A fluorescent label according to claim 7, wherein said fluorescent aromatic dimer comprises a phenanthridinium monomer.

10. A fluorescent label according to claim 9, wherein said fluorescent aromatic dimer is an ethidium dimer.

11. A fluorescent label according to claim 7, wherein said fluorescent aromatic dimer comprises an acridinium monomer.

12. A fluorescent label according to claim 7, wherein said double stranded DNA comprises a single stranded DNA chain of at least about 8 nucleotides at the 5′

or 3′

terminus.

13. A fluorescent label according to claim 12, wherein said double stranded DNA comprises a single stranded DNA chain of at least about 8 nucleotides as a loop.

18. A composition comprising:

(a) a double stranded DNA of at least about 20 base pairs and

(b) intercalated and non-covalently bound in said double stranded DNA at least one fluorescent molecule of at least two positive charges, having at least one fluorescent monomeric unit and having a binding affinity to double stranded DNA of at least about 5×

10⁶ M<sup>−

1</sup> and up to about 1 fluorescent unit per 4 nucleotides in a nucleic acid strand, wherein said fluorescent unit is characterized by having at least one aromatic ring per monomeric unit, wherein said double stranded DNA is covalently linked to a polypeptide or saccharide.