Compositions and methods for RNA hybridization applications

Patent metadata

Field	Value
Publication number	US-9303287-B2
Application number	US-200913203137-A
Country	US
Kind code	B2
Filing date	Dec 2, 2009
Priority date	Feb 26, 2009
Publication date	Apr 5, 2016
Grant date	Apr 5, 2016

Abstract

The invention provides methods and compositions for hybridizing at least one molecule to an RNA target. The invention may, for example, eliminate the use of, or reduce the dependence on formamide in RNA hybridization applications. Compositions for use in the invention include an aqueous composition comprising at least one nucleic acid sequence and at least one polar aprotic solvent in an amount effective to enable hybridization to RNA.

First claim

The invention claimed is:

1. A composition comprising at least one polar aprotic solvent in an amount effective to enable hybridization of a nucleic acid sequence to an RNA sequence within a cell in a sample having a preserved cell morphology, and at least 10% dextran sulfate, wherein the polar aprotic solvent is not dimethyl sulfoxide (DMSO).

2. The composition according to claim 1 , wherein the concentration of polar aprotic solvent is about 1% to 90% (v/v).

3. The composition according to claim 2 , wherein the concentration of polar aprotic solvent is 5% to 10% (v/v).

4. The composition according to claim 2 , wherein the concentration of polar aprotic solvent is 10% to 20% (v/v).

5. The composition according to claim 2 , wherein the concentration of polar aprotic solvent is 20% to 30% (v/v).

6. The composition according to claim 1 , wherein the polar aprotic solvent is non-toxic.

7. The composition according to claim 1 , with the proviso that the composition does not contain formamide.

8. The composition according to claim 6 , with the proviso that the composition contains less than 10% formamide.

9. The composition according to claim 8 , with the proviso that the composition contains less than 2% formamide.

10. The composition according to claim 9 , with the proviso that the composition contains less than 1% formamide.

11. The composition according to claim 1 , wherein the polar aprotic solvent has lactone, sulfone, nitrile, sulfite, and/or carbonate functionality.

12. The composition according to claim 1 , wherein the polar aprotic solvent has a dispersion solubility parameter between 17.7 to 22.0 MPa 1/2 , a polar solubility parameter between 13 to 23 MPa 1/2 , and a hydrogen bonding solubility parameter between 3 to 13 MPa 1/2 .

13. The composition according to claim 1 , wherein the polar aprotic solvent has a cyclic base structure.

14. The composition according to claim 1 , wherein the polar aprotic solvent is selected from the group consisting of:

where X is O and R1 is alkyldiyl, and

where X is optional and if present, is chosen from O or S,
where Z is optional and if present, is chosen from O or S,
where A and B independently are O or N or S or part of the alkyldiyl or a primary amine,
where R is alkyldiyl, and
where Y is O or S or C, and wherein if Y is C, then either X or Z is not present or both X and Z are not present.

15. The composition according to claim 1 , wherein the polar aprotic solvent is selected from the group consisting of: acetanilide, acetonitrile, N-acetyl pyrrolidone, 4-amino pyridine, benzamide, benzimidazole, 1,2,3-benzotriazole, butadienedioxide, 2,3-butylene carbonate, γ-butyrolactone, caprolactone (epsilon), chloro maleic anhydride, 2-chlorocyclohexanone, chloroethylene carbonate, chloronitromethane, citraconic anhydride, crotonlactone, 5-cyano-2-thiouracil, cyclopropylnitrile, dimethyl sulfate, dimethyl sulfone, 1,3-dimethyl-5-tetrazole, 1,5-dimethyl tetrazole, 1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone, epsilon-caprolactam, ethanesulfonylchloride, ethyl ethyl phosphinate, N-ethyl tetrazole, ethylene carbonate, ethylene trithiocarbonate, ethylene glycol sulfate, glycol sulfite, furfural, 2-furonitrile, 2-imidazole, isatin, isoxazole, malononitrile, 4-methoxy benzonitrile, 1-methoxy-2-nitrobenzene, methyl alpha bromo tetronate, 1-methyl imidazole, N-methyl imidazole, 3-methyl isoxazole, N-methyl morpholine-N-oxide, methyl phenyl sulfone, N-methyl pyrrolidinone, methyl sulfolane, methyl-4-toluenesulfonate, 3-nitroaniline, nitrobenzimidazole, 2-nitrofuran, 1-nitroso-2-pyrrolidinone, 2-nitrothiophene, 2-oxazolidinone, 9,10-phenanthrenequinone, N-phenyl sydnone, phthalic anhydride, picolinonitrile (2-cyanopyridine), 1,3-propane sultone, β-propiolactone, propylene carbonate, 4H-pyran-4-thione, 4H-pyran-4-one (γ-pyrone), pyridazine, 2-pyrrolidone, saccharin, succinonitrile, sulfanilamide, sulfolane, 2,2,6,6-tetrachlorocyclohexanone, tetrahydrothiapyran oxide, tetramethylene sulfone (sulfolane), thiazole, 2-thiouracil, 3,3,3-trichloro propene, 1,1,2-trichloro propene, 1,2,3-trichloro propene, trimethylene sulfide-dioxide, and trimethylene sulfite.

16. The composition according to claim 1 , wherein the polar aprotic solvent is selected from the group consisting of:

17. The composition according to claim 1 , wherein the polar aprotic solvent is:

18. The composition according to claim 1 , further comprising at least one additional component selected from the group consisting of: buffering agents, salts, accelerating agents, chelating agents, detergents, and blocking agents.

19. The composition according to claim 18 , wherein the salt is NaCl and/or the buffering agent is phosphate buffer.

20. The composition according to claim 19 , wherein the dextran sulfate is present at a concentration of 10% to 40%, the NaCl is present at a concentration of 0 mM to 1200 mM, and/or the phosphate buffer is present at a concentration of 0 mM to 50 mM.

21. The composition according to claim 20 , wherein the dextran sulfate is present at a concentration of 10% to 30%, the NaCl is present at a concentration of 300 mM to 600 mM, and/or the phosphate buffer is present at a concentration of 5 mM to 20 mM.

22. The composition according to claim 18 , wherein the accelerating agent is selected from the group consisting of: formamide, DMSO, glycerol, propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol, glycol, and 1,3 propanediol, and the buffering agent is citric acid buffer.

23. The composition according to claim 22 , wherein the formamide is present at a concentration of 0.1-5%, the DMSO is present at a concentration of 0.01% to 10%, the glycerol, propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol, glycol, and 1,3 propanediol are present at a concentration of 0.1% to 10%, and the citric acid buffer is present at a concentration of 1 mM to 50 mM.

24. The composition according to claim 18 , wherein the blocking agent is selected from the group consisting of: total human DNA, herring sperm DNA, salmon sperm DNA, and calf thymus DNA.

25. The composition according to claim 24 , wherein the total human DNA, herring sperm DNA, salmon sperm DNA, and calf thymus DNA are present at a concentration of 0.01 to 10 μg/μL.

26. The composition according to claim 1 , comprising 40% of at least one polar aprotic solvent, 10% dextran sulfate, 300 mM NaCl, and 5 mM phosphate buffer.

27. The composition according to claim 1 , comprising 15% of at least one polar aprotic solvent, 20% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, and 0.1 μg/μl total human DNA.

28. The composition according to claim 1 , comprising 15% of at least one polar aprotic solvent, 20% dextran sulfate, 600 mM NaCl, 10 mM citric acid buffer pH 6.2, and 0.1 μg/μL herring sperm DNA, or salmon sperm DNA, or calf thymus DNA, or 0.5% formamide, or 1% ethylene glycol, or 1% 1,3 propanediol.

Assignees

• Matthiesen Steen Hauge
• Dako Denmark As

Inventors

• Matthiesen Steen Hauge

Classifications

• C12Q1/6832 Primary

  Enhancement of hybridisation reaction · CPC title

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title

• C12Q1/6841 Primary

  In situ hybridisation · CPC title

• C12Q2527/137

  Concentration of a component of medium · CPC title