Aza-benzazolium containing cyanine dyes

• US 8,883,415 B2
• Filed: 03/04/2013
• Issued: 11/11/2014
• Est. Priority Date: 04/30/1999
• Status: Expired due to Fees

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Haugland, Richard, Yue, Stephen
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/784,413
• Publication Number:
  US 20130178395A1
• Time in Patent Office: 617 Days
• Field of Search: 435/6.1, 435/29, 435/34, 435/91.2
• US Class Current: 435/6.1
• CPC Class Codes: C09B 23/02 the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
• 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 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 2001/302 Stain compositions
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/531 Production of immunochemical test materials
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Unsymmetrical cyanine dyes that incorporate an aza-benzazolium ring moiety are described, including cyanine dyes substituted by a cationic side chain, monomeric and dimeric cyanine dyes, chemically reactive cyanine dyes, and conjugates of cyanine dyes. The subject dyes are virtually non-fluorescent when diluted in aqueous solution, but exhibit bright fluorescence when associated with nucleic acid polymers such as DNA or RNA, or when associated with detergent-complexed proteins. A variety of applications are described for detection and quantitation of nucleic acids and detergent-complexed proteins in a variety of samples, including solutions, electrophoretic gels, cells, and microorganisms.

Claims

1. A method for detecting double-stranded DNA in the presence of single-stranded nucleic acid in a sample, wherein the method comprises the steps:

a) combining the sample with a staining solution, wherein the staining solution comprises one or more dye compounds having a structure according to

2. The method according to claim 1, wherein X is O, n is 0, R² is methyl, and R⁶ and R⁷ taken together form a fused aromatic ring -—

R¹¹═

R¹²—

R¹³═

R¹⁴—

wherein R¹¹, R¹², R¹³, and R¹⁴ are independently selected from the group consisting of hydrogen, C₁-C₆ alkyl, and —

OR⁸.

3. The method according to claim 1, wherein R⁵ is a CYCLIC SUBSTITUENT that is an aryl group and R⁴ is a C₁-C₆ alkyl group.

4. The method according to claim 1, wherein R⁵ is a C₁-C₆ alkyl group and R⁴ is a CYCLIC SUBSTITUENT that is an aryl group.

5. The method according to claim 1, wherein the dye is substituted by at least one TAIL substituent.

6. The method according to claim 5, wherein the TAIL is according to formula LINK-SPACER-CAP;

wherein LINK is a single covalent bond, an ether linkage (—

O—

), a thioether linkage (—

S—

), or an amine linkage (—

NR²⁰—

);

where R²⁰ is hydrogen, a C₁-C₈ alkyl group or SPACER-CAP;

SPACER is a covalent linkage; and

CAP is —

OR²¹, —

SR²¹, —

NR²¹R²², or —

NR²¹R²²R²³;

where R²¹, R²², and R²³ are independently selected from the group consisting of hydrogen, a C₁-C₈ alkyl, and a C₁-C₈ cycloalkyl wherein the alkyl or cycloalkyl are optionally substituted by one or more substituents selected from the group consisting of halogen, hydroxyl, a C₁-C₈ alkoxy, amino, carboxy, sulfo, and phenyl, where the phenyl is optionally substituted by one or more substituents selected from the group consisting of halogen, hydroxyl, a C₁-C₈ alkoxy, amino, a C₁-C₈ aminoalkyl, a C₁-C₈ sulfoalkyl, and a C₁-C₈ carboxyalkyl;

or one or more R²¹, R²², and R²³, taken in combination with R²⁰ and SPACER, or with SPACER alone, forms a 5- or 6-membered ring.

7. The method according to claim 6, wherein R⁴ or R⁵ is independently a TAIL or a CYCLIC SUBSTITUENT substituted by TAIL.

8. The method according to claim 7, wherein R⁴ is a TAIL and R⁵ is a C₁-C₆ alkyl group.

9. The method according to claim 8, wherein CAP is —

NR²¹R²², where R²¹ and R²² are independently C₁-C₆ alkyl groups.

10. The method according to claim 8, wherein CAP is —

NR²¹R²²R²³, where ²¹R²², and R²³ are independently C₁-C₆ alkyl groups.

11. The method according to claim 7, wherein R⁵ is a CYCLIC SUBSTITUENT that is an aryl or heteroaryl and R⁴ is a TAIL, where CAP is —

OR²¹ or —

SR²¹.

12. The method according to claim 7, wherein R⁵ is a TAIL.

13. The method according to claim 12, wherein CAP is —

NR²¹R²²R²³, where R²¹, R²² and R²³ are independently C₁-C₆ alkyl groups.

14. The method according to claim 1, wherein the staining solution comprises one or more dyes having the structure of Compound 15, Compound 16, Compound 22, Compound 23, Compound 24, or Compound 25.

15. The method according to claim 1, wherein the double stranded DNA is in solution, immobilized on a solid or semi-solid matrix, or present in a biological structure.

16. The method according to claim 1, further comprising quantifying the double-stranded DNA present in the sample.

17. The method according to claim 1, wherein the fluorescence of the dye when associated with double-stranded DNA is distinguishable from the fluorescence of the dye when associated with single-stranded nucleic acid.

18. The method according to claim 15, wherein the biological structure is a biological cell or portion thereof, virus particle or tissue section.

19. The method according to claim 15, wherein the double-stranded DNA is in solution that is free from cells or portions thereof.

20. The method according to claim 15, wherein the solid or semi-solid matrix is a polymeric gel, an array, a glass slide, a polymeric microparticle, a membrane a silica-based support, or a plastic-based support.