Thermally Responsive Conjugates

Abstract

A method of amplifying a target nucleic acid in a DNA sample comprising (a) contacting the DNA sample containing the target nucleic acid with a DNA polymerase, at least two oligonucleotide primers designed to flank the target nucleic acid, a mixture of dATP, dGTP, dCTP, and dTTP, and a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature sensitive polymer, (b) heating the output of step (a) to a temperature at which the conjugate precipitates and thus the DNA polymerase is no longer inhibited and (c) amplifying the target nucleic acid, e.g. by performing PCR steps of denaturing the target nucleic acid, annealing the primers to the target nucleic acid, and extending the primers, wherein step (c) is repeated at least two times. Also provided is a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature sensitive polymer and kits and aqueous compositions comprising the same.

Claims

1. A method of amplifying a target nucleic acid in a DNA sample comprising: (a) contacting the DNA sample containing the target nucleic acid with: (i) a DNA polymerase, (ii) at least two oligonucleotide primers designed to flank the target nucleic acid, (iii) a mixture of dATP, dGTP, dCTP, and dTTP, and (iv) a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature sensitive polymer, (b) heating the output of step (a) to a temperature at which the conjugate precipitates and thus the DNA polymerase is no longer inhibited, (c) amplifying the target nucleic acid, e.g. by performing PCR steps of denaturing the target nucleic acid, annealing the primers to the target nucleic acid, and extending the primers wherein step (c) is repeated at least two times.

2. The method of claim 1, wherein the DNA polymerase inhibitor is vidarabine, lamivudine, rifamycin SV monosodium salt, neobavaisoflavone, hexaprenylhydroquinone, abacavir hemisulfate, aphidicolin, mithramycin A, tenofovir or thiolutin.

3. The method of claim 1, wherein the DNA polymerase inhibitor is Zidovudine.

4. The method of any of claims 1-3, wherein the negative temperature sensitive polymer is selected from: (i) Poly(N-substituted acrylamide)s and its derivatives, (ii) Poly(methyl vinyl ether), (iii) Poly(N-vinylcaprolactam), (iv) Poly(2-substituted 2-oxazoline)s and its derivatives, (v) Poly(2-substituted 2-oxazine)s and its derivatives, (vi) Hydroxypropylmethyl cellulose, (vii) Ethylhydroxyethyl cellulose, (viii) Hydroxyethyl cellulose, (ix) Poly(asparagine)s and its derivatives, and (x) PNiPMAM.

5. The method of any of claims 1-4, wherein the conjugate precipitates at a temperature between 35° C. and 65° C., preferably between 40° C. and 60° C., more preferably between 45° C. and 55° C.

6. The method of any of claims 1-5, wherein the conjugate precipitates at a temperature between 48° C. and 52° C.

7. The method of claim 5 or 6, wherein 2° C. above the precipitation temperature, 10% or less of the conjugate is in solution and 2° C. below the precipitation temperature, 90% or more of the conjugate is in solution.

8. The method of claim 7, wherein 1° C. above the precipitation temperature, 10% or less of the conjugate is in solution and 1° C. below the precipitation temperature, 90% or more of the conjugate is in solution.

9. The method of any preceding claim, wherein the conjugate comprises the DNA polymerase inhibitor covalently attached at or near a terminus of the negative temperature sensitive polymer.

10. The method of claim 9, wherein the conjugate comprises the DNA polymerase inhibitor covalently attached at a terminus of the negative temperature sensitive polymer.

11. The method of any preceding claim, wherein the DNA polymerase is a heat-stable DNA polymerase.

12. The method of claim 11, wherein the DNA polymerase is Taq polymerase.

13. A kit comprising (a) a DNA polymerase, and (b) a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature sensitive polymer, wherein the conjugate precipitates at a temperature between 35° C. and 65° C., preferably between 40° C. and 60° C., more preferably between 45° C. and 55° C.

14. The kit of claim 13, wherein the conjugate precipitates at a temperature between 48° C. and 52° C.

15. The kit of any of claims 13-14, wherein the DNA polymerase inhibitor is vidarabine, lamivudine, rifamycin SV monosodium salt, neobavaisoflavone, hexaprenylhydroquinone, abacavir hemisulfate, aphidicolin, mithramycin A, tenofovir or thiolutin.

16. The kit of any of claims 13-14, wherein the DNA polymerase inhibitor is Zidovudine.

17. The kit of any of claims 13-16, wherein 2° C. above the precipitation temperature, 10% or less of the conjugate is in solution and 2° C. below the precipitation temperature, 90% or more of the conjugate is in solution.

18. The kit of claim 17, wherein 1° C. above the precipitation temperature, 10% or less of the conjugate is in solution and 1° C. below the precipitation temperature, 90% or more of the conjugate is in solution.

19. The kit of any of claims 13-18, wherein the conjugate comprises the DNA polymerase inhibitor covalently attached at or near a terminus of the negative temperature sensitive polymer.

20. The kit of claim 19, wherein the conjugate comprises the DNA polymerase inhibitor covalently attached at a terminus of the negative temperature sensitive polymer.

21. The kit of any of claims 13-20, wherein the DNA polymerase is a heat-stable DNA polymerase.

22. The kit of claim 21, wherein the DNA polymerase is Taq polymerase.

23. An aqueous composition comprising (i) a DNA polymerase bound to (ii) a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature sensitive polymer.

24. The aqueous composition of claim 23, wherein the conjugate precipitates at a temperature between 35° C. and 65° C., preferably between 40° C. and 60° C., more preferably between 45° C. and 55° C.

25. The aqueous composition of claim 24, wherein the conjugate precipitates at a temperature between 48° C. and 52° C.

26. The aqueous composition of any of claims 23-25, wherein the DNA polymerase inhibitor is vidarabine, lamivudine, rifamycin SV monosodium salt, neobavaisoflavone, hexaprenylhydroquinone, abacavir hemisulfate, aphidicolin, mithramycin A, tenofovir or thiolutin.

27. The aqueous composition of any of claims 23-25, wherein the DNA polymerase inhibitor is Zidovudine.

28. The aqueous composition of any of claims 24-27, wherein 2° C. above the precipitation temperature, 10% or less of the conjugate is in solution and 2° C. below the precipitation temperature, 90% or more of the conjugate is in solution.

29. The aqueous composition of claim 28, wherein 1° C. above the precipitation temperature, 10% or less of the conjugate is in solution and 1° C. below the precipitation temperature, 90% or more of the conjugate is in solution.

30. The aqueous composition of any of claims 23-29, wherein the conjugate comprises the DNA polymerase inhibitor covalently attached at or near a terminus of the negative temperature sensitive polymer.

31. The aqueous composition of claim 30, wherein the conjugate comprises the DNA polymerase inhibitor covalently attached at a terminus of the negative temperature sensitive polymer.

32. The aqueous composition of any of claims 23-31, wherein the DNA polymerase is a heat-stable DNA polymerase.

33. The aqueous composition of claim 32, wherein the DNA polymerase is Taq polymerase.

34. A conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature sensitive polymer.

35. The conjugate of claim 34, wherein the conjugate precipitates at a temperature between 35° C. and 65° C., preferably between 40° C. and 60° C., more preferably between 45° C. and 55° C.

36. The conjugate of claim 35, wherein the conjugate precipitates at a temperature between 48° C. and 52° C.

37. The conjugate of any of claims 34-36, wherein the DNA polymerase inhibitor is vidarabine, lamivudine, rifamycin SV monosodium salt, neobavaisoflavone, hexaprenylhydroquinone, abacavir hemisulfate, aphidicolin, mithramycin A, tenofovir or thiolutin.

38. The conjugate of any of claims 34-36, wherein the DNA polymerase inhibitor is Zidovudine.

39. The conjugate of any of claims 35-38, wherein 2° C. above the precipitation temperature, 10% or less of the conjugate is in solution and 2° C. below the precipitation temperature, 90% or more of the conjugate is in solution.

40. The conjugate of claim 39, wherein 1° C. above the precipitation temperature, 10% or less of the conjugate is in solution and 1° C. below the precipitation temperature, 90% or more of the conjugate is in solution.

41. The conjugate of any of claims 34-40, wherein the conjugate comprises the DNA polymerase inhibitor covalently attached at or near a terminus of the negative temperature sensitive polymer.

42. The conjugate of claim 41, wherein the conjugate comprises the DNA polymerase inhibitor covalently attached at a terminus of the negative temperature sensitive polymer.

Description

EXAMPLES

[0082] The invention is now further described in specific examples with reference to the accompanying drawings in which:

[0083] FIG. 1 shows quantification of cDNA from a RTase reaction performed at different temperatures, with RTase alone or conjugated to zidovudine-poly-N-isopropylmethacrylamide; RTase cDNA was subsequently amplified and quantified by qPCR.

Example 1

Preparation of Thermo-Responsive zidovudine-poly-N-isopropylmethacrylamide Inhibitor

Step 1: Preparation of Polymerisable Zidovudine by Reaction With Propargyl Acrylate

[0084] Propargyl acrylate (413 μL, 2 eq.) was added to ethanol (30 ml) in a sealable vessel and flushed with nitrogen for 5 minutes before zidovudine (0.50 g), copper sulfate (30 mg in water, 1 mol %) and sodium ascorbate (62 mg in water, 17 mol %) were added. The copper sulfate and sodium ascorbate can be added as a solid if enough water is added to the reaction to dissolve them both. The vessel was sealed and heated to 30° C. with gentle stirring and left overnight. The solvent was then partially evaporated using nitrogen gas (enough for a column, about 4 hours). Flash silica chromatography (100% ethyl acetate) was then used for purification and solvent was removed under reduced pressure at room temperature. A white solid of 0.332 g, (0.88 mmol, 47%) yield) was obtained.

Step 2: Free-Radical Polymerisation and Polymer Purification

[0085] In a glass vial, 26 mg polymerizable zidovudine, 50 mg of 1,1′-azobis(cyclohexanecarbonitrile) and 0.9 g N-isopropylmethacrylamide were dissolved using 20 ml of ethanol. The mixture was then bubbled with nitrogen for 5 min, sealed with a screw cap and placed in an oven at 70° C. for 24 hours. Polymer “P” was prepared as described above. Polymer “P1X” was prepared as above but the addition of 26 mg polymerizable zidovudine was done after 2 hours polymerisation. Polymer “P2X” was prepared as polymer “P1X” but adding 52 mg polymerizable zidovudine instead of 26 mg. After polymerisation all polymers were purified using the same protocol. For this, the polymerisation mixture was added to ca. 150 ml deionised water and the initial precipitate removed by filtration and discarded. The filter flow-through was then heated to 60° C. The precipitate formed was removed by filtration using a glass fibre membrane with a nominal pore size of 1.2 μm. After collection, the membrane was cooled to room temperature and the thermo-responsive polymer eluted with ethanol/water 50/50% (v/v). The obtained polymer had a lower critical solution temperature of approximately 52° C. as observed by formation of turbidity upon heating a polymer solution dispersed in water.

Example 2

[0086] Reverse Transcriptase (RTase) Reversible Inhibition Experiment Using Thermo-Responsive zidovudine-poly-N-isopropylmethacrylamide Inhibitor From Example 1

[0087] This example demonstrates the use of the thermo-responsive zidovudine-poly-N-isopropylmethacrylamide inhibitor from Example 1 for reversible inhibition of RTase.

[0088] The reaction was performed using the UltraScript 2.0 Reverse Transcriptase from PCR Bio (UK), according to the instructions of the manufacturer. General conditions were, for a 20 μl total reaction volume:

[0089] RNA=5 μg template plus random hexamer primers, in PCR Bio buffer mix.

[0090] PCR Bio RTase, 324 nM.

[0091] 150 ng Zidovudine-poly-N-isopropylmethacrylamide.

RTase Reaction

[0092] The reaction was carried out at a range of temperatures below and above the LCST of the zidovudine-poly-N-isopropylmethacrylamide, to assess both inhibition and thermal reversibility of the conjugate. Individual reaction vials were kept at the set temperature for 30 min and the reaction allowed to proceed. The temperatures used were 25° C., 43° C., 55° C. and 35° C., the latter being done after the 55° C. step. After each incubation, 1-2 μl of cDNA product was taken for qPCR amplification and quantification. The results are presented in FIG. 1. Inhibition detected by observing loss of product peak.

[0093] Note that the zidovudine-poly-N-isopropylmethacrylamide prepared in Example 1 had a lower critical solution temperature of approximately 52° C., thus at temperatures above approximately 52° C., substantially all of the conjugate was out of solution.

[0094] This confirmed the dependence of temperature on RTase activity (availability) when RTase was conjugated to the thermally responsive polymer and that inhibition was reversible.