Dimeric and trimeric nucleic acid dyes, and associated systems and methods

• US 20060211028A1
• Filed: 03/16/2006
• Published: 09/21/2006
• 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 7,776,567 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 method of determining nucleic acid formation or a lack thereof in a sample that may or may not comprise a target nucleic acid, the method comprising:

providing a test solution comprising the sample and a fluorescent nucleic acid dye, the fluorescent nucleic acid dye having the formula;

wherein BRIDGE is a substantially aliphatic, substantially neutral linker comprising from about 8 to about 150 non-hydrogen atoms;

Q₁ is a dye constituent selected from a fluorescent nucleic acid dye constituent, a non-fluorescent nucleic acid dye constituent, a fluorescent non-nucleic acid dye constituent, and a non-fluorescent non-nucleic acid dye constituent;

Q₂ is a dye constituent selected from a fluorescent nucleic acid dye constituent, a non-fluorescent nucleic acid dye constituent, a fluorescent non-nucleic acid dye constituent, and a non-fluorescent non-nucleic acid dye constituent;

at least one dye constituent of the Q₁ dye constituent and the Q₂ dye constituent is a reporter dye constituent;

at least one dye constituent of the Q₁ dye constituent and the Q₂ dye constituent is a fluorescent nucleic acid dye constituent or a non-fluorescent nucleic acid dye constituent; and

the reporter dye constituent and the fluorescent nucleic acid dye constituent are optionally the same;

performing a process using the test solution that would be sufficient for amplification of the target nucleic acid should the sample comprise the target nucleic acid; and

illuminating the test solution with light at a wavelength sufficient for absorption by the reporter dye constituent and determining fluorescent emission or a lack thereof.

2. A method of determining nucleic acid formation or a lack thereof in a sample that may or may not comprise a target nucleic acid, the method comprising:

providing a test solution comprising the sample and a fluorescent nucleic acid dye, the fluorescent nucleic acid dye having the formula;

wherein BRIDGE is a substantially aliphatic, substantially neutral linker comprising from about 15 to about 150 non-hydrogen atoms;

Q₁ is a dye constituent selected from a fluorescent nucleic acid dye constituent, a non-fluorescent nucleic acid dye constituent, a fluorescent non-nucleic acid dye constituent, and a non-fluorescent non-nucleic acid dye constituent;

Q₂ is a dye constituent selected from a fluorescent nucleic acid dye constituent, a non-fluorescent nucleic acid dye constituent, a fluorescent non-nucleic acid dye constituent, and a non-fluorescent non-nucleic acid dye constituent;

Q₃ is a dye constituent selected from a fluorescent nucleic acid dye constituent, a non-fluorescent nucleic acid dye constituent, a fluorescent non-nucleic acid dye constituent, and a non-fluorescent non-nucleic acid dye constituent;

at least one dye constituent of the Q₁ dye constituent, the Q₂ dye constituent, and the Q₃ dye constituent is a reporter dye constituent;

at least one dye constituent of the Q₁ dye constituent, the Q₂ dye constituent, and the Q₃ dye constituent is a fluorescent nucleic acid dye constituent or a non-fluorescent nucleic acid dye constituent; and

the reporter dye constituent and the fluorescent nucleic acid dye constituent are optionally the same;

performing a process using the test solution that would be sufficient for amplification of the target nucleic acid should the sample comprise the target nucleic acid; and

illuminating the test solution with light at a wavelength sufficient for absorption by the reporter dye constituent and determining fluorescent emission or a lack thereof.

3. The method of any of claims 1 and 2, wherein the fluorescent nucleic acid dye constituent is selected from an acridine dye, an asymmetric cyanine dye, a symmetric cyanine dye, a phenanthridinium dye, and a pyronin dye, and a styryl dye.

4. The method of claim 1, wherein said performing comprises performing a real-time PCR process.

5. The method of claim 1, wherein at least one dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

6. The method of claim 5, wherein each R₁ is H;

R₂ is H;

R₃ represents where BRIDGE attaches to the structure;

each R₆ is —

CH₃; and

each R₇ is —

CH₃.

7. The method of claim 1, wherein at least one dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

8. The method of claim 1, wherein at least one dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

9. The method of claim 1, wherein at least one dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

10. The method of claim 1, wherein BRIDGE has the formula:

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)_ι

-]_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;

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 ι

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

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

11. The method of claim 10, wherein BRIDGE comprises from about 8 to about 100 non-hydrogen atoms, inclusive.

12. The method of claim 10, wherein each of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰, independently, is a NABEG comprising a moiety that comprises at least one bond linkage that comprises at least one amide bond, urethane bond, urea bond, thiourea bond, ether bond, or thioether bond;

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

13. The method of claim 1, wherein BRIDGE has the formula:

—

(CH₂)_x—

C(═

O)NH—

(CH₂)_α

—

[O—

(CH₂)_β

]_b—

[O—

(CH₂)_γ

]_c—

NH(O═

C)—

(CH₂)_x—

wherein each x, independently, is an integer selected from 1 to 11, inclusive;

α

is an integer selected from 2 to about 20, inclusive;

each of β and

γ

, independently, is 2 or 3;

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

c is zero or 1.

14. The method of claim 13, wherein x is 5;

α and

γ

are the same and are 2 or 3;

β

is 2;

b is 0, 1, 2, or 3; and

c is 1.

15. The method of claim 13, wherein Q₁ and Q₂ are the same.

16. The method of claim 13, wherein each dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

17. The method of claim 13, wherein each dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

18. The method of claim 13, wherein each dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

19. The method of claim 13, wherein each dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

20. The method of any of claims 1 and 4, wherein the fluorescent nucleic acid dye has the structure:

21. A composition having the formula:

22. The composition of claim 21, wherein BRIDGE comprises from about 12 to about 60 non-hydrogen atoms, inclusive.

23. The composition of claim 21, wherein BRIDGE comprises from about 15 to about 40 non-hydrogen atoms, inclusive.

24. The composition of claim 21, wherein Q₁ is a fluorescent nucleic acid dye constituent selected from an acridine dye, an asymmetric cyanine dye, a symmetric cyanine dye, a phenanthridinium dye, and a pyronin dye, and a styryl dye, and Q₂ is a fluorescent nucleic acid dye constituent selected from an acridine dye, an asymmetric cyanine dye, a symmetric cyanine dye, a phenanthridinium dye, and a pyronin dye, and a styryl dye.

25. The composition of claim 24, wherein the acridine dye has the structure:

26. The composition of claim 25, wherein each R₁ is H;

R₂ is H;

R₃ represents where BRIDGE attaches to the structure;

each R₆ is —

CH₃; and

each R₇ is —

CH₃.

27. The composition of claim 24, wherein the asymmetric cyanine dye has the structure:

28. The composition of claim 24, wherein the asymmetric cyanine dye the structure:

29. The composition of claim 24, wherein the phenanthridinium dye has the structure:

30. The composition of claim 21, wherein BRIDGE has the formula:

—

(CH₂)_x—

C(═

O)NH—

(CH₂)_α

—

[O—

(CH₂)_β

]_b—

[O—

(CH₂)_γ

]_c—

NH(O═

C)—

(CH₂)_x—

wherein each x, independently, is an integer selected from 1 to 11, inclusive;

α

is an integer selected from 2 to about 20, inclusive;

each of β and

γ

, independently, is 2 or 3;

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

c is zero or 1.

31. The composition of claim 30, wherein x is 5;

α and

γ

are the same and are 2 or 3;

β

is 2;

b is 0, 1, 2, or 3; and

c is 1.

32. The composition of claim 30, wherein Q₁ and Q₂ are the same.

33. The composition of claim 30, wherein each dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

34. The composition of claim 30, wherein each dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

35. The composition of claim 30, wherein each dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

36. The composition of claim 30, wherein each dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

37. A dye having the structure:

38. A dye having the structure:

39. A dye having the structure:

40. A dye having the structure:

41. A composition having the formula:

42. The composition of claim 41, 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)_ι

-]_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 ι

, 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;

wherein T is a substituted carbon, a substituted nitrogen, or an aryl optionally comprising at least one hetero atom selected from O, N and S;

L₃ is covalently bound to Q₃ and 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; and

B′

has the formula;

—

(CH₂)<sub>α

′</sub>

—

[A¹¹—

(CH₂)<sub>β

′</sub>

—

]_a′

[A¹²—

(CH₂)<sub>γ

′</sub>

—

]_b′

[A¹³ —

(CH₂)<sub>δ

′</sub>

—

]_c′

A¹⁴—

wherein —

(CH₂)<sub>α

′</sub>

is covalently linked to T and A¹⁴ is covalently linked to L₃;

each of A¹¹, A¹², A¹³, and A¹⁴ is independently 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

′

, and δ

′

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

each of a′

, b′

, and c′

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

43. A composition having the formula:

44. The composition of claim 43, 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)_ι

-]_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-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 ι

, 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;

wherein T is a substituted carbon, a substituted nitrogen, or an aryl optionally comprising at least one hetero atom selected from O, N and S;

L₃ is covalently bound to R_r and 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; and

B′

has the formula;

—

(CH₂)<sub>α

′</sub>

—

[A¹¹—

(CH₂)<sub>β

′</sub>

—

]_a′

[A¹²—

(CH₂)<sub>γ

′</sub>

—

]_b′

[A¹³—

(CH₂)<sub>δ

′</sub>

—

]_c′

A¹⁴-,

wherein —

(CH₂)<sub>α

′</sub>

is covalently linked to T and A¹⁴ is covalently linked to L₃;

each of A¹¹, A¹², A¹³, and A¹⁴ is independently 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

′

, and δ

′

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

each of a′

, b′

, and c′

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

45. A method of using a dye, comprising:

conjugating the composition of claim 44 to a substrate molecule.

46. The method of claim 45, wherein the substrate molecule 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, a virus, and a molecule of a solid surface.

47. A method of preparing a sample that may or may not comprise nucleic acid, comprising:

providing a combination of the sample and the composition of any of claims 21, 30 and 41, wherein if nucleic acid is present in the sample, a nucleic acid-dye complex is formed.

48. The method of claim 47, further comprising incubating the combination.

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

providing a combination of the sample and the composition of any of claims 21, 30 and 41, wherein if nucleic acid is present in the sample, a nucleic acid-dye complex is formed; and

illuminating the combination with light at a wavelength sufficient such that if a nucleic acid-complex is formed the light is absorbed thereby, and determining fluorescent emission or a lack thereof.

50. A kit for determining nucleic acid formation or a lack thereof in a sample that may or may not comprise a target nucleic acid, the kit comprising:

at least one composition sufficient for amplification of the target nucleic acid in the sample should the sample comprise the target nucleic acid; and

a composition of any of claims 21, 30 and 41.