EMBODIMENTS OF A PROBE AND METHOD FOR TARGETING NUCLEIC ACIDS

• US 20140220573A1
• Filed: 07/19/2012
• Published: 08/07/2014
• Est. Priority Date: 07/19/2011
• Status: Active Grant

• Current Assignee: University of Idaho
• Original Assignee: University of Idaho
• Inventors: Hrdlicka, Patrick Jerzy
• Granted Patent: US 9,885,082 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• 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: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6879 for sex determination
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Disclosed embodiments concern a probe comprising one or more pairs of monomers capable of targeting a nucleic acid target. The pair of monomers may be arranged in a manner that promotes thermoinstability of the probe complex, thus producing a probe capable of locating and/or detecting a target. The probe also may comprise one or more natural or non-natural nucleotides capable of Watson-Crick base pairing with an isosequential nucleic acid target. Particular disclosed embodiments concern a method of using the disclosed probe to target nucleic acids. In particular disclosed embodiments, the probe may be incubated with a target nucleic acid and then be detected.

Claims

1. A double stranded probe, comprising:

a pair of monomers comprising a first monomer having a formula

2. The probe according to claim 1 wherein the heteroatom-containing moiety is selected from ether (R^aOR^b), hydroxyl (R^aOH), silyl ether (R^aR^bR^cSiOR^d), phosphine (PR^aR^bR^c), thiol (R^aSH), thioether/sulfide (R^aSR^b), disulfide (R^aSSR^b), isothiocyanate (R^aNCS), isocyanate (R^aNCO), amine (NH₂, NHR^a, NR^aR^b), amide (R^aNR^bC(O)R^c), ester (R^aOC(O)R^b), halogen (I, Br, Cl, F), carbonate (R^aOC(O)OR^b), carboxyl (R^aC(O)OH), carboxylate (R^aCOO⁻

), ester (R^aC(O)OR^b), ketone (R^aC(O)R^b), phosphate (R^aOP(O)OH₂), phosphoryl (R^aP(O)(OH)₂), sulfinyl (R^aS(O)R^b), sulfonyl (R^aSO₂R^b), carbonothioyl (R^aC(S)R^b or R^aC(S)H), sulfino (R^aS(O)OH), sulfo (R^aSO₃H), amide (R^aC(O)NR^bR^cC), azide (N₃), nitrile (R^aCN), isonitrile (R^aN⁺C⁻

), and nitro (R^aNO₂);

R^a represents the remaining monomer structure, which is attached to the abovementioned functional groups at the position indicated for R¹; and

R^b, R^c, and R^d independently are hydrogen, aliphatic, aryl, heteroaliphatic, heteroaryl, and any combination thereof.

3. The probe according to claim 1 wherein R² and R³ independently are selected from a heteroatom functional group comprising phosphorous, sulfur, nitrogen, oxygen, selenium, and/or a metal.

4. The probe according to claim 1 wherein R² and R³ independently are selected from a phosphate group of a natural nucleotide, non-natural nucleotide, non-nucleosidic linker, or combinations thereof.

5. The probe according to claim 1 wherein R² and R³ independently have a formula

6. The probe according to claim 1 wherein R² and R³ independently are

9. The probe according to claim 1 wherein R⁴ is selected from adenine, guanine, cytosine, uracil, thymine, 2-thiouracil, 2,6-diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimdine-2-(3H)-one, or any derivative thereof.

11. The probe according to claim 1 wherein the intercalator is selected from pyrene, coronene, perylene, anthracene, naphthalene, a porphyrin, a nucleobase, a metal chelator, azapyrene, thiazole orange, ethidium, an indole, a pyrrole, a benzimidizole, or functionalized derivatives thereof.

16. The probe according to claim 1 wherein either the first monomer or the second monomer has a formula

17. The probe according to claim 1 wherein either the first monomer or the second monomer has a formula

20. The probe according to claim 1 wherein either the first monomer or the second monomer has any one of the following formulas:

22. The probe according to claim 1 wherein the natural nucleotide is selected from adenine, guanine, cytosine, uracil, or thymine;

and the non-natural nucleobase is selected from a C-5 functionalized pyrimidine, a C6-functionalized pyrimidine, a C7-functionalized 7-deazapurine, a C8-functionalized purine, 2,6-diaminopurine, 2-thiouracil, 4-thiouracil, deoxyinosine, or 3-(2′

-deoxy-β

-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimdine-2-(3H)-one.

25. The probe according to claim 1 having a formula

5′

(B₁B₂ . . . B_m)(XA)(B₁B₂ . . . B_n)(XA)_f(B₁B₂ . . . B_o)(XA)_g(B₁B₂ . . . B_p)(XA)_h(B₁B₂ . . . B_q)(XA)_i(B₁B₂ . . . B_r)(XA)_j(B₁B₂ . . . B_s)

3′

(C₁C₂ . . . C_m)(DP)(C₁C₂ . . . C_n)(DP)_f(C₁C₂ . . . C_o)(DP)_g(C₁C₂ . . . C_p)(DP)_h(C₁C₂ . . . C_q)(DP)_i(C₁C₂ . . . C_r)(DP)_j(C₁C₂ . . . C_s)

wherein B₁, B₂ and B_m-s may be any natural, non-natural nucleotide, or a non-nucleosidic linker, wherein m-r ranges from zero to about 28;

f, g, h, i and j range from zero to 10;

X is the first monomer;

A is the complement Watson-Crick base pairing nucleotide of P;

C is any natural or non-natural nucleotide capable of Watson-Crick base pairing with any one of B₁, B₂ and B_m-s;

P is the second monomer, and D is the complement Watson-Crick base pairing nucleotide of X.

26. The probe according to claim 1 wherein the probe is selected to recognize a predetermined sequence of a single-stranded or double-stranded nucleic acid target.

28. The probe according to claim 1 further comprising one or more bulge monomers selected from

33. The probe according to claim 1 wherein the first monomer and the second monomer are arranged in a +n or −

n zipper orientation, wherein n ranges from 0 to about 10.

36. The probe according to claim 1 further comprising a signal generating moiety capable of being detected and wherein the signal generating moiety is selected from a fluorophore, a member of a specific binding pair, a nanoparticle, or combinations thereof.

39. The probe according to claim 1 further comprising a secondary entity that facilitates cell-uptake;

a quencher;

a crosslinking reagent capable of forming bonds between the probe and nucleic acids, proteins, sugars, lipids or other biomolecules;

a nucleic acid cargo moiety selected from single-stranded DNA, single-stranded RNA, double-stranded DNA, double-stranded RNA, plasmid, or gene;

or combinations thereof.

40. The probe according to claim 1 wherein the probe is used in solution, on a solid surface, or in combination with a colloidal material.

43. The probe according to claim 1 wherein the probe is selected from any one of SEQ ID Nos. 5-10, 12-33, 38-65, 72-85, 94-105, 112-129, 136-218, 220-239, or 246-253.

7-8. -8. (canceled)

10. (canceled)

12-15. -15. (canceled)

18-19. -19. (canceled)

21. (canceled)

23-24. -24. (canceled)

27. (canceled)

29-32. -32. (canceled)

34-35. -35. (canceled)

37-38. -38. (canceled)

41-42. -42. (canceled)

44. A method for associating a probe with a target, comprising:

selecting a double stranded probe comprising a monomer having a formula

45. The method according to claim 44 wherein the probe further comprises a second monomer having a formula selected from

47. The method according to claim 44 wherein the nucleic acid target comprises one or more polypurine units.

50. The method according to claim 44 wherein the nucleic acid target is a mixed-sequence, hairpin nucleic acid target.

51. The method according to claim 44 wherein the nucleic acid target is isosequential with the probe.

55. The method according to claim 44 wherein the probe-target complex is detected by fluorescence spectroscopy, electrophoresis, absorption spectroscopy, flow cytometry, and combinations thereof.

65. The method according to claim 44 wherein the probe is used for gender determination in mammals selected from ungulates or ruminants.

69. The method according to claim 65 wherein the mammals are bovines, equines or porcines.

70. The method according to claim 44 wherein the target is selected from a stem of a molecular beacon;

a target region embedded within PCR amplicons;

a target region embedded within circular or linearized plasmids;

a target region embedded within genomic DNA;

a target region embedded within microorganisms;

a nucleic acid sequence associated with B cell and T cell leukemias, lymphomas, breast cancer, colon cancer, and neurological cancers;

a virus or other microorganism wherein the probe is used to detect and/or identify the microorganism;

a genome of an oncogenic or pathogenic virus;

a bacterium;

an intracellular parasite selected from Plasmodium species, Leishmania (sp.), Cryptosporidium parvum, Entamoeba histolytica, Giardia lamblia, Toxoplasma, Eimeria, Theileria, and Babesia;

a second insulin promoter;

a PPAR gamma promoter;

or a CEBP promoter.

46. (canceled)

48-49. -49. (canceled)

52-54. -54. (canceled)

56-64. -64. (canceled)

66-68. -68. (canceled)

71-77. -77. (canceled)

78. A kit, comprising:

a double stranded nucleic acid probe comprising a pair of monomers comprising a first monomer having a formula

79-86. -86. (canceled)