METHODS AND DEVICES FOR DE NOVO OLIGONUCLEIC ACID ASSEMBLY

• US 20160229884A1
• Filed: 02/03/2016
• Published: 08/11/2016
• Est. Priority Date: 02/04/2015
• Status: Active Grant

• Current Assignee: Twist Bioscience Corporation
• Original Assignee: Twist Bioscience Corporation
• Inventors: Marsh, Eugene P., Peck, Bill J., Indermuhle, Pierre F., Fernandez, Andres, Banyai, William
• Granted Patent: US 10,669,304 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00711 Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07H 1/00 Processes for the preparation of sugar derivatives
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C23C 16/455 characterised by the method used for introducing gases into reaction chamber or for modifying gas flows in reaction chamber

Abstract

Methods and devices are provided herein for surfaces for de novo nucleic acid synthesis which provide for low error rates. In addition, methods and devices are provided herein for increased nucleic acid mass yield resulting from de novo nucleic acid synthesis.

Claims

1. A method for preparing a surface for oligonucleic acid synthesis, the method comprising:

(a) providing a silicon wafer comprising a surface;

(b) depositing a first molecule on the surface at a first region, wherein the first molecule binds to the surface and lacks a reactive group that binds to a nucleoside phosphoramidite;

(c) depositing a second molecule on the surface at a second region, wherein the second region comprises a plurality of loci, wherein each locus is surrounded by the first region, and wherein the second molecule binds to the surface and lacks a reactive group that binds to a nucleoside phosphoramidite; and

(d) depositing a mixture on the surface at the second region, wherein the mixture comprises the second molecule and a third molecule, wherein the third molecule binds to the surface and is capable of binding nucleoside phosphoramidite, and wherein the second molecule and the third molecule are present in the mixture in a molar ratio of 10;

1 to about 2500;

1.

2. The method of claim 1, wherein the second molecule and the third molecule both have a higher surface energy than a surface energy of the first molecule.

3. The method of claim 2, wherein a difference in water contact angle between the first region and the second region is at least 10 degrees.

4. The method of claim 1, wherein the third molecule is a silane.

5. The method of claim 1, wherein the third molecule is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxy silane, 3-glycidoxypropyltrimethoxysilane, 3-iodo-propyltrimethoxysilane, or octylchlorosilane.

7. The method of claim 4, wherein the silane is an aminosilane.

8. The method of claim 1, wherein the second molecule is propyltrimethoxysilane.

9. The method of claim 1, wherein the first molecule is a fluorosilane.

10. The method of claim 9, wherein the fluorosilane is (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane.

11. The method of claim 1, wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of 50:

1 to 2500;

1.

13. The method of claim 1, wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of 2000:

1.

14. The method of claim 1, wherein the first molecule or the second molecule lacks a free hydroxyl, amino, or carboxyl group.

16. The method of claim 1, wherein the mixture or the first molecule is in a gaseous state when deposited on the surface.

17. The method of claim 1, wherein the surface comprises a layer of silicon dioxide.

18. The method of claim 1, comprising:

coating the surface with a light-sensitive material;

exposing the first region of the surface to a light source to remove a portion of the light-sensitive material coated on the surface;

depositing the first molecule on the surface;

removing a remaining portion of the light-sensitive material coated on the surface to expose the second region;

depositing the second molecule on the surface at the second region; and

depositing the mixture on the surface at the second region.

36. The method of claim 18, further comprising applying oxygen plasma to the surface prior to coating the surface with the light-sensitive material or after exposing predetermined regions of the surface to light.

6. (canceled)

12. (canceled)

15. (canceled)

19-35. -35. (canceled)

37. A method for oligonucleic acid synthesis, the method comprising:

(a) providing predetermined sequences for at least 20,000 non-identical oligonucleic acids;

(b) providing a silicon wafer comprising a patterned surface, wherein the patterned surface is generated by;

depositing a first molecule on the surface at a first region, wherein the first region comprises a plurality of loci, and wherein the first molecule binds to the surface and lacks a reactive group that binds to a nucleoside phosphoramidite; and

depositing a mixture on the surface at the first region, wherein the mixture comprises the first molecule and a second molecule, wherein the second molecule binds to the surface and is capable of binding nucleoside phosphoramidite, and wherein the first molecule and the second molecule are present in the mixture in a molar ratio of 10;

1 to about 2500;

1; and

(c) synthesizing the at least 20,000 non-identical oligonucleic acids each at least 10 bases in length, wherein the at least 20,000 non-identical oligonucleic acids encode sequences with an aggregate deletion error rate of less than 1 in 1500 bases compared to the predetermined sequences, and wherein each of the at least 20,000 non-identical oligonucleic acids extends from a different locus.

38. The method of claim 37, further comprising depositing a third molecule on the surface at a second region, wherein the third molecule binds to the surface and lacks a reactive group that binds to nucleoside phosphoramidite, wherein the first molecule and the second molecule both have a higher surface energy than a surface energy of the third molecule.

39. The method of claim 38, wherein a difference in water contact angle between the first region and the second region is at least 10 degrees.

40. The method of claim 37, wherein the second molecule is a silane.

41. The method of claim 37, wherein the second molecule is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxy silane, 3-glycidoxypropyltrimethoxysilane, 3-iodo-propyltrimethoxysilane, or octylchlorosilane.

42. The method of claim 40, wherein the silane is an aminosilane.

44. The method of claim 37, wherein the first molecule is propyltrimethoxysilane.

45. The method of claim 38, wherein the third molecule is a fluorosilane.

46. The method of claim 45, wherein the fluorosilane is (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane.

47. The method of claim 37, wherein the mixture comprises the first molecule and the second molecule present in a molar ratio of 50:

1 to 2500;

1.

49. The method of claim 37, wherein the mixture comprises the first molecule and second present in a molar ratio of 2000:

1.

55. The method of claim 37, wherein each of the at least 20,000 non-identical oligonucleic acids is 10 bases to 1 kb in length.

56. The method of claim 55, wherein each of the at least 20,000 non-identical oligonucleic acids is about 50 to about 120 bases in length.

57. The method of claim 37, wherein the aggregate deletion error rate of the at least 20,000 non-identical oligonucleic acids is less than about 1 in 1700 bases compared to the predetermined sequences.

58. The method of claim 62, wherein the aggregate deletion error rate of the assembled at least 200 preselected nucleic acids is achieved without correcting errors.

62. The method of claim 37, further comprising:

(d) releasing the at least 20,000 non-identical oligonucleic acids from the patterned surface;

(e) suspending the at least 20,000 non-identical oligonucleic acids in a solution; and

(f) subjecting the solution comprising at least 20,000 non-identical oligonucleic acids to a polymerase chain assembly reaction to assemble at least 200 preselected nucleic acids, wherein the assembled at least 200 preselected nucleic acids encode sequences with an aggregate deletion error rate of less than 1 in 1500 bases compared to the predetermined sequences.

43. (canceled)

48. (canceled)

50-54. -54. (canceled)

59-61. -61. (canceled)

63-82. -82. (canceled)

83. A device for oligonucleic acid synthesis, the device comprising:

(a) a silicon wafer having a surface;

(b) a plurality of recesses or protrusions on the surface, wherein each recess or protrusion comprises;

(i) a width distance that is 6.8 nm to 500 nm,

(ii) a pitch distance that is about twice the width distance, and

(iii) a depth distance that is about 60% to about 125% of the pitch distance; and

(c) a plurality of loci on the surface, wherein each locus has a diameter of 0.5 to 100 μ

m, and wherein each locus comprises at least two of the plurality of recesses or protrusions.

88. The device of claim 83, wherein each locus has a diameter of 0.5 to 50 μ

m.

93. The device of claim 83, wherein each locus comprises a molecule that binds to the surface and a nucleoside phosphoramidite.

94. The device of claim 93, wherein the molecule that binds to the surface and the nucleoside phosphoramidite is a silane.

95. The device of claim 93, wherein the molecule that binds to the surface and the nucleoside phosphoramidite is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-iodo-propyltrimethoxysilane, or octylchlorosilane.

97. The device of claim 94, wherein the silane is an aminosilane.

98. The device of claim 83, wherein a region surrounding each locus comprises a molecule that binds to the surface and lacks a nucleoside phosphoramidite.

99. The device of claim 98, wherein the molecule that binds to the surface and lacks the nucleoside phosphoramidite is a fluorosilane.

100. The device of claim 99, wherein the fluorosilane is (tridecafluoro-1, 1,2,2-tetrahydrooctyl)trichlorosilane.

102. A method for oligonucleic acid synthesis, comprising:

(a) providing predetermined sequences;

(b) providing the device of claim 83;

(c) synthesizing a plurality of non-identical oligonucleic acids at least 10 bases in length, wherein each of the non-identical oligonucleic acids extends from a different locus.

108. The method of claim 102, comprising:

(d) releasing the plurality of non-identical oligonucleic acids from the surface;

(e) suspending the plurality of non-identical oligonucleic acids in a solution; and

(f) subjecting the solution comprising the plurality of non-identical oligonucleic acids to a polymerase chain assembly reaction to assemble at least 200 preselected nucleic acids, wherein the assembled at least 200 preselected nucleic acids encode sequences with an aggregate deletion error rate of less than 1 in 1500 bases compared to the predetermined sequences.

113. The device of claim 83, further comprising a plurality of clusters on the surface, wherein each of the clusters comprises a subset of the plurality of loci, and wherein the subset of the plurality of loci comprises 50 to 500 loci.

114. The device of claim 113, wherein each of the clusters has a diameter of 0.5 to 2 mm.

84-87. -87. (canceled)

89-92. -92. (canceled)

96. (canceled)

101. (canceled)

103-107. -107. (canceled)

109-112. -112. (canceled)