Pressure cycling reactor and methods of controlling reactions using pressure

• US 6,036,923 A
• Filed: 10/28/1997
• Issued: 03/14/2000
• Est. Priority Date: 03/07/1995
• Status: Expired due to Term

• Current Assignee: Pressure BioSciences, Inc.
• Original Assignee: BioSeq, Inc. (Pressure BioSciences, Inc.)
• Inventors: Green, David J., Laugharn, James A. Jr., Dreier, Gustav H., Rudd, Edwin A.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Siew, Jeffrey
• Application Number:
  US08/793,213
• Time in Patent Office: 868 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 422/33, 422/39, 422/82.13, 422/99, 422/102, 422/103, 422/129, 422/239, 422/242, 436/94, 536/23.1
• US Class Current: 422/82.13
• CPC Class Codes: B01J 19/185 of the pulsating type
• CPC Class Codes: B01J 2219/00162 controlling the pressure
• CPC Class Codes: B01J 2219/002 inside the reactor
• CPC Class Codes: B01J 2219/00211 comparing a sensed parameter with a pre-set value
• CPC Class Codes: B01J 2219/00229 of the reaction system
• CPC Class Codes: B01J 3/00 Processes of utilising sub-atmospheric or super-atmospheric pressure to effect chemical or physical change of matter; Apparatus therefor pressure vessels for containing or storing compressed, liquefied or solidified gases F17C
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Methods and apparatus in which pressure provides precise control over the timing and preferably synchronization of chemical reactions, particularly enzymatic reactions.

Claims

1. A reactor for intermittently inhibiting activity of a sample by controlling pressure applied to the sample, comprising:

a fluid reservoir for containing a test reagent,

a reactor body defining a chamber for receiving a sample, and

a system for providing flow of the test reagent into said chamber while said chamber is pressurized, said system comprising;

a first valve located in a first conduit in communication with said fluid reservoir,

a second valve located in a second conduit in communication with said chamber,

a first pressurizer located between said fluid reservoir and said chamber and in communication with said first conduit and said second conduit, and

a third valve associated with said first pressurizer for venting said first pressurizer,

with said first valve in an open position and said second valve in a closed position, said reservoir being in communication with said first pressurizer to allow fluid flow to said first pressurizer;

with said first valve in a closed position, said second valve in an open position, and said third valve in a closed position, said pressurizer being in communication with said chamber to pressurize said chamber;

with said first valve in a closed position, said second valve in an open position, and said third valve cycled between an open position and a closed position, said pressure in said chamber being pulsed between an activity-inhibiting pressure P₁ and an activity-allowing pressure P₂.

2. The reactor of claim 1 further providing flow of the test material out of said chamber, said system further comprising:

a fourth valve located in a third conduit in communication with said chamber,

a fifth valve located in a fourth conduit downstream of said third conduit,

a second pressurizer located downstream of said chamber and in communication with said third conduit and said fourth conduit, and

a sixth valve associated with said second pressurizer for venting said second pressurizer,

with said first valve in a closed position, said second valve in an open position, said third valve in a closed position, and said fourth valve in a closed position, said pressurizer being in communication with said chamber to pressurize said chamber;

with said first valve in a closed position, said second valve in an open position, said third valve cycled between an open position and a closed position, and said fourth valve in a closed position said pressure in said chamber being pulsed; and

with said first valve in a closed position, said second valve in an open position, said third valve in a closed position, said fourth valve in an open position, said fifth valve in a closed position, and said sixth valve in an open position said first pressurizer being in communication with said second pressurizer to enable flow through from the first pressurizer through the chamber to the second pressurizer.

3. A method of controlling an enzymatic reaction, comprising

(i) providing a sample mixture in a sample vessel at reversibly inactivating pressure P₁, the sample mixture comprising an enzyme;

(ii) exposing the sample mixture to activating pressure P₂ ;

(iii) maintaining the activating pressure P₂ for time period t₁ ; and

(iv) exposing the sample mixture to inactivating pressure P₃, thereby controlling an enzymatic reaction.

4. A method of claim 3, in which the inactivating pressure P₁ is P_i,x in step (i), at which pressure an enzymatic reaction step is reversibly inhibited;

the activating pressure P₂ is P_a,y in steps (ii) and (iii), at which pressure the enzymatic reaction step can occur, the exposing step (iii) comprising changing the pressure to P_a,y in a length of time γ

t_a,y ; and

the inactivating pressure P₃ is P_i,z in step (iv), at which an additional enzymatic reaction step is reversibly inhibited, the exposing step (iv) comprising changing the pressure to P_i,z in a length of time γ

t_i,z, ;

x being an integer greater than or equal to zero, y being an integer greater than or equal to 1, and z being an integer greater than or equal to 1, thereby controlling the enzymatic reaction.

5. A method of claim 3 in which the sample mixture of step (i) further comprises a substrate of the enzyme.

6. A method of claim 3, in which t₁ corresponds to the average length of a single enzymatic event.

7. A method of claim 3 in which the enzyme has distributive properties.

8. A method of claim 3 in which the enzyme has processive properties.

9. A method of claim 3, in which the substrate is immobilized within the sample vessel, further comprising after step (iv) the step of removing a component of the sample mixture from the sample vessel while maintaining the inactivating pressure in the sample vessel, or the step of adding a liquid to the sample mixture while maintaining the inactivating pressure in the sample vessel.

10. A method of claim 9 in which the removed component is selected from the group consisting of a restriction endonuclease, a restriction endonuclease cleavage product, a exonuclease, a nucleotide, and a combination thereof.

11. A method of claim 9 in which the component passes through a semi-permeable material when the component is removed from the sample vessel.

12. A method of claim 3, further comprising after step (iv) the step of detecting a characteristic of a component of the sample mixture.

13. A method of claim 12 in which the characteristic is selected from the group consisting of radioactivity, fluorescence, chemiluminescence, molecular ion charge/mass ratio, electrochemical potential, light emission, surface plasmon resonance, and infra-red absorption.

14. A method of claim 6 in which the substrate is a nucleic acid;

and the enzyme is a restriction endonuclease, whereby at least one cleavage fragment is cleaved from the nucleic acid substrate.

15. A method of claim 6 in which the substrate is a nucleic acid immobilized within the sample vessel;

and the enzyme is an exonuclease, whereby at least one nucleotide is cleaved from the nucleic acid substrate.

16. A method of claim 15 in which the nucleic acid is selected from the group consisting of double stranded DNA, single stranded DNA, DNA containing both double and single stranded regions, and RNA.

17. A method of claim 5 in which the substrate is a first substrate, and further comprising a second substrate, wherein the enzyme acts to attach the first substrate to the second substrate.

18. A method of claim 17 in which the first substrate is a nucleotide;

the second substrate comprises an RNA oligonucleotide or a DNA nucleotide; and

the enzyme is a polyribonucleotide phosphorylase where the second substrate is an RNA oligonucleotide and a terminal transferase where the second substrate is a DNA nucleotide.

19. A method of claim 17 in which the first substrate is a nucleotide;

the second substrate comprises an RNA or DNA oligonucleotide; and

the enzyme is a transferase selected from the enzyme class 2.7.7.

20. A method of claim 5 in which the substrate is a compound with a chiral or pro-chiral functional group;

and the enzyme acts on the substrate enantiospecifically.

21. A method of claim 20 in which the enzyme is a protease, a dehydrogenase, an oxidase, a transferase, a lipase, or an esterase.

22. A method of claim 3 wherein the enzyme in steps (i)-(iv) is a first enzyme, and the sample mixture in steps (i)-(iv) is a first sample mixture, further comprising after step (iv) the following steps (v)-(vi )

(v) providing a second sample mixture in a sample vessel, the sample mixture comprising a second enzyme at a reversibly inactivating pressure P_i,j, the second enzyme being the same as or different from the first enzyme in steps (i)-(iv), and the sample vessel being the same as the sample vessel in steps (i)-(iv) or being a second sample vessel connected to the first sample vessel by a valve;

(vi) exposing the second sample mixture to a reversibly activating pressure, thereby controlling enzymatic reaction steps of the first and second enzymes.

23. A method of claim 22 in which the first and second enzymes are the same enzyme.

24. A method of claim 23 in which the substrate is immobilized within the sample vessel and the second enzyme is different from the first enzyme, further comprising between steps (iv) and (v) the step of removing the first enzyme from the sample vessel while maintaining the inactivating pressure in the sample vessel by eluting with an eluting solution.

25. A method of claim 24 in which the substrate is a double stranded nucleic acid, the first enzyme is a 5'"'"'-3'"'"' exonuclease, and the second enzyme is a 3'"'"'-5'"'"' exonuclease, thereby identifying one or more nucleotides by sequencing with the first enzyme and confirming the one or more nucleotides by sequencing with the second enzyme.

26. A method of claim 4 in which the sample mixture at pressure P_i,x is at temperature T_i,x, whereby the enzyme is inhibited;

the sample mixture at pressure P_a,y is at temperature T_a,y, whereby the enzyme is active; and

the sample mixture at pressure P_i,z is at temperature T_i,z, whereby the enzyme is inhibited;

each of T_i,z and T_a,y being independently the same as or different from T_i,x.

27. A method of claim 4 in which each of δ

t_a,y and δ

t_i,z is between 10 and 250 milliseconds.

28. A method of claim 4 in which the sum (δ

t_a,y +t_a,y +δ

t_i,z) is less than or equal to 1000 milliseconds.

29. A method of claim 3 in which steps (ii)-(iv) are one cycle, further comprising the steps of repeating the cycle of steps (ii)-(iv) at least 49 times, wherein the value for each respective value of P_i,x, P_i,z, δ

t_a,y, P_a,y, and δ

t_i,z in a cycle, is independent of the respective value in any other cycle.

30. A method of claim 22 in which steps (v) and (vi) are one cycle, further comprising the steps of repeating the cycle of steps (v) and (vi) at least 49 times.

31. A method of claim 3 further comprising the step of adding a fluid to the sample mixture while maintaining the pressure of the sample mixture.

32. A method of claim 3 further comprising the step of removing a component of the sample mixture from the sample vessel while maintaining the pressure of the sample mixture.

54. The method of claim 3 further comprising, between steps (i) and (ii), introducing a substrate of the enzyme into the sample vessel while reversibly inactivating pressure is maintained.

55. The method of claim 3 wherein the sample mixture of step (i) further comprises a substrate;

and the method further comprises, between steps (i) and (ii), introducing into the sample vessel a component necessary for reaction between the enzyme and substrate, while reversibly inactivating pressure is maintained.

56. The method of claim 5, wherein the initial temperature of the sample mixture is unsuitable for reaction of the enzyme with the substrate;

and the method further comprises, between steps (i) and (ii), changing the temperature of the sample mixture to a temperature suitable for reaction of the enzyme with the substrate.

33. A method of affecting the thermodynamic equilibrium of a reaction comprising the conversion of at least one starting material to at least one product, said method comprising

(i) providing a sample mixture comprising said at least one starting material in a sample vessel, the sample mixture being at a pressure P₁ and a temperature T₀ ;

(ii) changing the sample mixture temperature to T₁ ;

(iii) increasing the pressure of the reaction mixture to P₂ in a length of time δ

t₁, wherein P₂ is at least 10,000 psi greater than P₁ ;

(iv) maintaining the pressure of the reaction mixture at P₂ for duration t₁ ; and

(v) reducing the pressure of the reaction mixture to P₃, thereby affecting the thermodynamic equilibrium of the reaction to convert said stating material to at least one product.

34. A method of claim 33, wherein t₁ is sufficient to allow the sample mixture to react.

35. A method of claim 33 in which the sample mixture includes a catalyst, further comprising after the pressure increasing step (iii) the step of allowing at least one product of said reaction to dissociate from the catalyst.

36. A method of claim 33, further comprising after step (iii) the step of removing a component of the sample mixture from the sample vessel.

37. A method for treating nucleic acid, comprising:

a) providing, in any order;

i) a sample vessel, ii) a nucleic acid substrate, iii) an enzyme capable of acting on the nucleic acid substrate, and iv) a pressurizer for controlling pressure in the vessel;

b) providing the enzyme and the nucleic acid, independently, in solution in the sample vessel while maintaining the enzyme under inactivating pressure P₁ ;

c) changing the pressure in the sample vessel to an enzyme activating pressure P₂ for a controlled period of time t₁, such that the enzyme is active and acts on the nucleic acid substrate for the time period;

then

d) changing the pressure to enzyme inactivating pressure P₃.

38. The method of claim 37, wherein the nucleic acid is double stranded DNA.

39. The method of claim 37, wherein the enzyme is an exonuclease.

40. The method of claim 39, wherein the exonuclease is Lambda exonuclease.

41. The method of claim 37, wherein the enzyme is a DNA polymerase or a RNA polymerase.

42. The method of claim 37 in which the enzyme has processive properties.

43. The method of claim 37, wherein the enzyme modifies the nucleic acid substrate.

44. The method of claim 43, further comprising the step of detecting a reaction product.

45. The method of claim 37, further comprising controlling reaction vessel temperature, wherein the inactivating pressure conditions include a temperature that permits a high level of enzyme activity when reaction vessel pressure is reduced to the enzyme activating pressure.

46. The method of claim 45 wherein the method further comprises maintaining the reaction vessel at a reversibly, enzyme inactivating temperature and an enzyme activating pressure, raising the pressure to an enzyme inactivating pressure, lowering the temperature to an enzyme activating temperature, and then lowering the pressure to an enzyme activating pressure.

47. The method of claim 46 comprising,

a) maintaining the enzyme at an inactivating temperature of less than approximately 5°

C., thereby rendering the enzyme substantially inactive;

b) adding the nucleic acid substrate to the inactive enzyme to create a reaction mixture;

c) increasing the pressure in the sample vessel to an enzyme inactivating pressure of greater than approximately 30,000 pounds per square inch;

d) raising the temperature of the reaction mixture in the sample vessel to greater than approximately 10°

C.;

e) for a controlled period of time, lowering the pressure in the sample vessel to an enzyme activating pressure of less than approximately 20,000 pounds per square inch, thereby rendering the enzyme active such that the enzyme acts on the nucleic acid substrate; and

f) raising the pressure in the sample vessel to an enzyme inactivating pressure.

48. The method of claim 47, wherein the enzyme activating pressure is a pressure of approximately 5,000 to 15,000 pounds per square inch.

49. The method of claim 47, wherein the enzyme activating temperature is approximately 15°

C. to 20°

C.

50. The method of claim 47, further comprising the step of

g) repeating a cycle of steps e and f) at least once.

51. The method of claim 50 in which the cycle of steps e) and f) is repeated at least five times.

52. The method of claim 37 in which the enzyme inactivating pressure is higher than the enzyme activating pressure.

53. The method of claim 37 in which the enzyme has distributive properties.