DYES AND LABELED MOLECULES

• US 20100317016A1
• Filed: 06/22/2010
• Published: 12/16/2010
• Est. Priority Date: 03/17/2005
• Status: Active Grant

• Current Assignee: Allelogic Biosciences Corporation, Biotium, Inc.
• Original Assignee: Allelogic Biosciences Corporation, Biotium, Inc.
• Inventors: Leung, Wai-Yee, Mao, Fei, Xin, Xing
• Granted Patent: US 8,530,195 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07D 209/08 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring
• CPC Class Codes: C07D 213/38 having only hydrogen or hydrocarbon radicals attached to the substituent nitrogen atom
• CPC Class Codes: C07D 221/10 Aza-phenanthrenes
• 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: C07D 491/147 the condensed system containing one ring with oxygen as ring hetero atom and two rings with nitrogen as ring hetero atom
• 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
• CPC Class Codes: C09B 15/00 Acridine dyes
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• 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 2563/173 staining/intercalating agent, e.g. ethidium bromide

Abstract

Dimeric and trimeric nucleic acid dyes, and associated systems and methods are provided. Such a dye may form a hairpin-like structure that enables it to stain nucleic acids via a release-on-demand mechanism, for example. Such a dye may have low background fluorescence in the absence of nucleic acids and high fluorescence in the presence of nucleic acids, upon binding therewith, for example. A dye provided herein may be useful in a variety of applications, such as in DNA quantitation in real-time PCR, for example.

Claims

1. A labeled biomolecule having a structure of

2. The labeled biomolecule according to claim 1, wherein BRIDGE is a substantially aliphatic linker comprising from about 15 to about 150 non-hydrogen atoms and up to one positive charge.

3. The labeled biomolecule of claim 2, wherein BRIDGE comprises:

-L₁-[A¹-(CH₂)_α

-]_a[A²-(CH₂)_β

-]_b[A³-(CH₂)_γ

-]_c[A⁴-(CH₂)_δ

-]_d[A⁵-(CH₂)_ε

-]_e[A⁶-(CH₂)_ζ

-]_f[A⁷-(CH₂)_η

-]_g[A⁸-(CH₂)_θ

-]_h[A⁹-(CH₂)_τ

-]_i-A¹⁰-L₂-

wherein each of L₁ and L₂, independently, is a moiety comprising a single bond;

a polymethylene unit having 1 carbon to about 12 carbons, inclusive, optionally comprising at least one hetero atom selected from N, O and S;

or an aryl optionally comprising at least one hetero atom selected from N, O and S;

L₁ is covalently bound to one dye constituent of the Q₁ dye constituent and the Q₂ dye constituent;

L₂ is covalently bound to the dye constituent of the Q₁ dye constituent and the Q₂ that is other than said one dye constituent;

each of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰, independently, is a nucleic-acid-binding-enhancing-group (NABEG);

a branched alkyl optionally comprising at least one hetero atom selected from N, O and S;

or at least one saturated 5- or 6-membered ring, optionally comprising at least one hetero atom selected from N, O and S;

each of α

, β

, γ

, ε

, ζ

, η

, θ

, and τ

, independently, is zero or an integer from 1 to about 20, inclusive;

each of a, b, c, d, e, f, g, h, and i, independently, is zero or an integer from 1 to about 20, inclusive; and

one of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ has the formula;

4. The labeled biomolecule according to claim 1, wherein B is selected from a nucleotide, an oligonucleotide, a peptide, a protein, a hapten, a drug, a microparticle, a synthetic polymer, a natural polymer, a biological cell, and a virus.

5. The labeled biomolecule according to claim 4, wherein B is a nucleotide or an oligonucleotide.

6. The labeled biomolecule according to claim 1, wherein at least one of Q₁ and Q₂ is selected from the group consisting of an acridine dye, an asymmetric cyanine dye, a symmetric cyanine dye, a phenanthridinium dye, a pyronin dye, and a styryl dye.

7. The labeled biomolecule according to claim 6, wherein Q₁ and Q₂ are each an asymmetric cyanine dye.

8. The labeled biomolecule according to claim 7, wherein Q₁ and Q₂ are each thiazole orange.

9. A method of determining presence or absence of nucleic acid in a sample, comprising:

(i) providing a reaction mixture comprising said sample and reagents necessary for amplifying nucleic acid;

(ii) subjecting said reaction mixture to a polymerization reaction under conditions suitable for formation of amplified nucleic acid;

(iii) adding a fluorescent nucleic acid dye, wherein said addition takes place before, during, or after step (ii), and wherein said dye has the formula;

10. The method of claim 9, wherein BRIDGE is a substantially aliphatic linker comprising from about 15 to about 150 non-hydrogen atoms and up to one positive charge.

11. The method of claim 10, wherein BRIDGE comprises:

-L₁-[A¹-(CH₂)_α

-][A²-(CH₂)_β

-]_b[A³-(CH₂)_γ

-]_c[A⁴-(CH₂)_δ

-]_d[A⁵-(CH₂)_ε

-]_e[A⁶-(CH₂)_ζ

-]_f[A⁷-(CH₂)_η

-]_g[A⁸-(CH₂)_θ

-]_h[A⁹-(CH₂)_τ

-]_i-A¹⁰-L₂-

wherein each of L₁ and L₂, independently, is a moiety comprising a single bond;

a polymethylene unit having 1 carbon to about 12 carbons, inclusive, optionally comprising at least one hetero atom selected from N, O and S;

or an aryl optionally comprising at least one hetero atom selected from N, O and S;

L₁ is covalently bound to one dye constituent of the Q₁ dye constituent and the Q₂ dye constituent;

L₂ is covalently bound to the dye constituent of the Q₁ dye constituent and the Q₂ that is other than said one dye constituent;

each of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰, independently, is a nucleic-acid-binding-enhancing-group (NABEG);

a branched alkyl optionally comprising at least one hetero atom selected from N, O and S;

or at least one saturated 5- or 6-membered ring, optionally comprising at least one hetero atom selected from N, O and S;

each of α

, β

, γ

, δ

, ε

, ζ

, η

, θ

, and τ

, independently, is zero or an integer from 1 to about 20, inclusive;

each of a, b, c, d, e, f, g, h, and i, independently, is zero or an integer from 1 to about 20, inclusive; and

one of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ has the formula;

12. The method of claim 9, wherein at least one of Q₁ and Q₂ is selected from the group consisting of an acridine dye, an asymmetric cyanine dye, a symmetric cyanine dye, a phenanthridinium dye, a pyronin dye, and a styryl dye.

13. The method of claim 12, wherein Q₁ and Q₂ are each an asymmetric cyanine dye.

14. The method of claim 13, wherein Q₁ and Q₂ are each thiazole orange.

15. A method of detecting the presence or absence of nucleic acid in a sample, comprising:

(i) mixing the sample suspected of containing said nucleic acid with a labeled biomolecule of claim 1, wherein the labeled biomolecule is a labeled nucleotide or labeled polynucleotide.

(ii) illuminating the reaction mixture with light; and

(iii) detecting fluorescence emission from said reaction mixture, wherein said emission is indicative of the presence of nucleic acid.

16. The method of claim 15, wherein BRIDGE is a substantially aliphatic linker comprising from about 15 to about 150 non-hydrogen atoms and up to one positive charge.

17. The method of claim 16, wherein BRIDGE comprises:

-L₁-[A¹-(CH₂)_α

-]_a[A²-(CH₂)_β

-]_b[A³-(CH₂)_γ

-]_c[A⁴-(CH₂)_δ

-]_d[A⁵-(CH₂)_ε

-]_e[A⁶-(CH₂)_ζ

-]_f[A⁷-(CH₂)_η

-]_g[A⁸-(CH₂)_θ

-]_h[A⁹-(CH₂)_τ

-]₁-A¹⁰-L₂-

wherein each of L₁ and L₂, independently, is a moiety comprising a single bond;

a polymethylene unit having 1 carbon to about 12 carbons, inclusive, optionally comprising at least one hetero atom selected from N, O and S;

or an aryl optionally comprising at least one hetero atom selected from N, O and S;

L₁ is covalently bound to one dye constituent of the Q₁ dye constituent and the Q₂ dye constituent;

L₂ is covalently bound to the dye constituent of the Q₁ dye constituent and the Q₂ that is other than said one dye constituent;

each of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰, independently, is a nucleic-acid-binding-enhancing-group (NABEG);

a branched alkyl optionally comprising at least one hetero atom selected from N, O and S;

or at least one saturated 5- or 6-membered ring, optionally comprising at least one hetero atom selected from N, O and S;

each of α

, β

, γ

, δ

, ε

, ζ

, η

, θ

, and τ

, independently, is zero or an integer from 1 to about 20, inclusive;

each of a, b, c, d, e, f, g, h, and i, independently, is zero or an integer from 1 to about 20, inclusive; and

one of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ has the formula;

18. The method of claim 15, wherein at least one of Q₁ and Q₂ is selected from the group consisting of an acridine dye, an asymmetric cyanine dye, a symmetric cyanine dye, a phenanthridinium dye, a pyronin dye, and a styryl dye.

19. The method of claim 18, wherein Q₁ and Q₂ are each an asymmetric cyanine dye.

20. The method of claim 19, wherein Q₁ and Q₂ are each thiazole orange.