Fluorescent Chemical Compounds Having High Selectivity for Double Stranded DNA, and Methods for Their Use

• US 20110217699A1
• Filed: 05/10/2011
• Published: 09/08/2011
• Est. Priority Date: 05/11/2005
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Huang, Shih Jung, Pinson, Patrick R., Bradford, Jolene A., Cheung, Ching-Ying, Yue, Stephen T.
• Application Number:
  US13/104,413
• Publication Number:
  US 20110217699A1
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: C07D 215/10 Quaternary compounds
• CPC Class Codes: C07D 215/227 only one oxygen atom which is attached in position 2
• CPC Class Codes: C07D 277/72 2-Mercaptobenzothiazole
• CPC Class Codes: C07D 277/74 Sulfur atoms substituted by carbon atoms
• CPC Class Codes: C07D 417/04 directly linked by a ring-member-to-ring-member bond
• 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: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 2201/06113 Coherent sources; lasers
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Chemical compounds having a high selectivity for double stranded DNA over RNA and single stranded DNA are disclosed. The chemical compounds are stains that become fluorescent upon illumination and interaction with double stranded DNA, but exhibit reduced or no fluorescence in the absence of double stranded DNA. The compounds can be used in a variety of biological applications to qualitatively or quantitatively assay DNA, even in the presence of RNA.

Claims

1. A chemical compound having the structure:

2. The chemical compound of claim 1, wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ are independently hydrogen, a hydroxyl group, an alkoxy group, a thiol, a thioalkyl, a thioaryl, a halogen, an alkyl group, an alkenyl group, an alkynyl group, an aromatic group, a primary amine group, a secondary amine group, a tertiary amine group, a reactive group, or combinations thereof.

3. The chemical compound of claim 1, wherein at least one of R¹, R², R³, and R⁴ comprises an aromatic group or an alkynyl group.

4. The chemical compound of claim 1, wherein R⁹ comprises an aromatic group, alkyl-aromatic, or an alkynyl group.

5. The chemical compound of claim 1, wherein R¹⁰ is an amine group.

6. The chemical compound of claim 1, wherein:

at least one of R¹, R², R³, and R⁴ comprises an aromatic group or an alkynyl group; and

R⁹ comprises an aromatic group, alkyl-aromatic group, or an alkynyl group.

7. The chemical compound of claim 1, wherein:

at least one of R¹, R², R³, and R⁴ comprises an aromatic group or an alkynyl group;

R⁹ comprises an aromatic group, alkyl-aromatic group, or an alkynyl group; and

R¹⁰ comprises an amine group.

8. The chemical compound of claim 1, wherein R¹² is a C₁-C₈ alkyl group.

9. The chemical compound of claim 1, wherein R¹² is a methyl group.

10. The chemical compound of claim 1, further comprising one or more cations or anions.

11. A kit comprising:

DNA, RNA, or both DNA and RNA; and

a chemical compound having the structure;

12. A method of detecting the presence or absence of double stranded DNA in a sample, the method comprising:

providing a sample suspected of containing double stranded DNA;

contacting the sample with a chemical compound to prepare a test sample;

illuminating the test sample with energy; and

detecting emission of energy from the test sample;

wherein the chemical compound has the structure;

13. The method of claim 12, further comprise calculating the concentration of double stranded DNA in the sample after the detecting step.

14. A chemical compound having the structure:

15. The chemical compound of claim 14, wherein:

R¹³_{, R}¹⁴_{, R}¹⁵_{, R}¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, and R²¹ are independently hydrogen, a hydroxyl group, an alkoxy group, a thiol, a thioalkyl, a thioaryl, a halogen, an alkyl group, an alkenyl group, an alkynyl group, an aromatic group, a primary amine group, a secondary amine group, a tertiary amine group, a reactive group, or combinations thereof.

16. The chemical compound of claim 14, wherein at least one of R¹³, R¹⁴, R¹⁵, and R¹⁶ comprises an aromatic group or an alkynyl group.

17. The chemical compound of claim 14, wherein R¹⁹ comprises an aromatic group, alkyl-aromatic, or an alkynyl group.

18. The chemical compound of claim 14, wherein R²⁰ is an amine group.

19. The chemical compound of claim 14, wherein:

at least one of R¹³, R¹⁴, R¹⁵, and R¹⁶ comprises an aromatic group or an alkynyl group; and

R¹⁹ comprises an aromatic group, alkyl-aromatic group, or an alkynyl group.

20. The chemical compound of claim 14, wherein:

at least one of R¹³, R¹⁴, R¹⁵, and R¹⁶ comprises an aromatic group or an alkynyl group;

R¹⁹ comprises an aromatic group, alkyl-aromatic group, or an alkynyl group; and

R²⁰ comprises an amine group.

21. The chemical compound of claim 14, wherein R²² is a C₁-C₈ alkyl group.

22. The chemical compound of claim 14, wherein R²² is a methyl group.

23. The chemical compound of claim 14, further comprising one or more cations or anions.

24. A kit comprising:

DNA, RNA, or both DNA and RNA; and

a chemical compound having the structure;

25. A method of detecting the presence or absence of double stranded DNA in a sample, the method comprising:

providing a sample suspected of containing double stranded DNA;

contacting the sample with a chemical compound to prepare a test sample;

illuminating the test sample with energy; and

detecting emission of energy from the test sample;

wherein the chemical compound has the structure;

26. The method of claim 25, further comprise calculating the concentration of double stranded DNA in the sample after the detecting step.