Design, Synthesis and Assembly of Synthetic Nucleic Acids

• US 20080300842A1
• Filed: 05/04/2005
• Published: 12/04/2008
• Est. Priority Date: 05/04/2004
• Status: Active Grant

• Current Assignee: DNA Twopointo Incorporated
• Original Assignee: DNA Twopointo Incorporated
• Inventors: Govindarajan, Sridhar, Ness, Jon E., Minshull, Jeremy S.
• Granted Patent: US 8,825,411 B2
• Time in Patent Office: Days
• US Class Current: 703/11
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1089 Design, preparation, screening or analysis of libraries using computer algorithms
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation

Abstract

Methods of synthesizing oligonucleotides with high coupling efficiency (>99.5%) are provided. Methods for purification of synthetic oligonucleotides are also provided. Instrumentation configurations for oligonucleotide synthesis are also provided. Methods of designing and synthesizing polynucleotides are also provided. Polynucleotide design is optimized for subsequent assembly from shorter oligonucleotides. Modifications of phosphoramidite chemistry to improve the subsequent assembly of polynucleotides are provided. The design process also incorporates codon biases into polynucleotides that favor expression in defined hosts. Design and assembly methods are also provided for the efficient synthesis of sets of polynucleotide variants. Software to automate the design and assembly process is also provided.

Claims

1-24. -24. (canceled)

25. A method of designing a polynucleotide, the method comprising:

a) selecting an initial polynucleotide sequence that codes for a polypeptide, wherein a codon frequency in said initial polynucleotide sequence is determined by a codon bias table; and

b) modifying an initial codon choice in said initial polynucleotide sequence in accordance with a design criterion, thereby constructing a final polynucleotide sequence that codes for said polypeptide.

26. The method of claim 25, wherein the design criterion comprises one or more of:

(i) exclusion of a restriction site sequence in said initial polynucleotide sequence;

(ii) incorporation of a restriction site sequence in said initial polynucleotide sequence;

(iii) a designation of a target G+C content in the initial polynucleotide sequence;

(iv) an allowable length of a sub-sequence that can be exactly repeated within either a 5′

-3′

or a 3′

-5′

strand of the initial polynucleotide sequence;

(v) an allowable annealing temperature of any sub-sequence to any other sub-sequence within either the 5′

-3′

or the 3′

-5′

strand of the initial polynucleotide sequence;

(vi) exclusion of a hairpin turn in the initial polynucleotide sequence;

(vii) exclusion of a repeat element in the initial polynucleotide sequence;

(viii) exclusion of a ribosome binding site in the initial polynucleotide sequence;

(ix) exclusion of a polyadenylation signal in the initial polynucleotide sequence;

(x) exclusion of a splice site in the initial polynucleotide sequence;

(xi) exclusion of an open reading frame in each possible 5′

reading frame in the initial polynucleotide sequence;

(xii) exclusion of a polynucleotide sequence that facilitates RNA degradation in the initial polynucleotide sequence;

(xiii) exclusion of an RNA polymerase termination signal in the initial polynucleotide sequence;

(xiv) exclusion of a transcriptional promoter in the initial polynucleotide sequence;

(xv) exclusion of an immunostimulatory sequence in the initial polynucleotide sequence;

(xvi) incorporation of an immunostimulatory sequence in the initial polynucleotide sequence;

(xvii) exclusion of an RNA methylation signal in the initial polynucleotide sequence;

(xviii) exclusion of a selenocysteine incorporation signal in the initial polynucleotide sequence;

(xix) exclusion of an RNA editing sequence in the initial polynucleotide sequence;

(xx) exclusion of an RNAi-targeted sequence in the initial polynucleotide sequence; and

(xxi) exclusion of an inverted repeat within the first 45 nucleotides encoding said polypeptide in the initial polynucleotide sequence.

27. The method of claim 25, wherein the design criterion comprises:

reduced sequence identity to a reference polynucleotide, and wherein

modifying said initial codon choice in said initial polynucleotide in accordance with said design criterion comprises altering a codon choice in said initial polynucleotide sequence to reduce sequence identity to said reference polynucleotide.

28. The method of claim 25, wherein the design criterion comprises:

increased sequence identity to a reference polynucleotide, and wherein

modifying said initial codon choice in said initial polynucleotide in accordance with said design criterion comprises altering a codon choice in said initial polynucleotide sequence to increase sequence identity to said reference polynucleotide.

29. A computer program product for use in conjunction with a computer system, the computer program product comprising a computer readable storage medium and a computer program mechanism embedded therein, the computer program mechanism comprising instructions for carrying out the method of claim 25.

33. A computer system comprising:

a central processing unit;

a memory, coupled to the central processing unit, the memory storing instructions for carrying out the method of claim 25.

30-32. -32. (canceled)

34-36. -36. (canceled)

37. A method of designing a set of oligonucleotides for assembly into a polynucleotide, the method comprising:

a) identifying a first plurality of single-stranded oligonucleotides that collectively encode all or a portion of a first strand of said polynucleotide, wherein each respective single-stranded oligonucleotide in said first plurality of single-stranded oligonucleotides is characterized by an annealing temperature to its exact complement that is in a first predetermined annealing temperature range;

b) identifying a second plurality of single-stranded oligonucleotides from said first plurality of single-stranded oligonucleotides, wherein a single-stranded oligonucleotide in said second plurality of single-stranded oligonucleotides is formed by joining an adjacent pair of oligonucleotides in said first plurality of single-stranded oligonucleotides;

c) identifying a third plurality of single-stranded oligonucleotides that collectively encode all or a portion of a second strand of said polynucleotide, wherein each respective single-stranded oligonucleotide in said third plurality of single-stranded oligonucleotides is characterized by an annealing temperature to its exact complement that is in a second predetermined annealing temperature range;

d) identifying a fourth plurality of single-stranded oligonucleotides from said third plurality of single-stranded oligonucleotides, wherein a single-stranded oligonucleotide in said fourth plurality of single-stranded oligonucleotides is formed by joining an adjacent pair of oligonucleotides in said third plurality of single-stranded oligonucleotides;

wherein said set of oligonucleotides comprises said second plurality of oligonucleotides and said fourth plurality of oligonucleotides; and

e) determining whether the set of oligonucleotides satisfies at least one assembly criterion, wherein

when the set of oligonucleotides satisfies said at least one assembly criterion, the set of oligonucleotides is selected; and

when the set of oligonucleotides does not satisfy said at least one assembly criterion, the set of oligonucleotides is rejected and steps a) through e) are repeated.

38. The method of claim 37, wherein a different first predetermined annealing temperature range and a different second predetermined annealing temperature range is used when steps a) through e) are repeated.

39. The method of claim 37, wherein said first predetermined annealing temperature range and said second predetermined annealing temperature range are the same.

40. The method of claim 37, wherein said first predetermined annealing temperature range and said second predetermined annealing temperature range are different.

41. The method of claim 37, wherein said first predetermined annealing temperature range and said second predetermined annealing temperature range is each between 45°

C. and 72°

C.

42. The method of claim 37, wherein said first predetermined annealing temperature range and said second predetermined annealing temperature range is each between 50°

C. and 65°

C.

43. The method of claim 37, wherein said first predetermined annealing temperature range and said second predetermined annealing temperature range is each between 55°

C. and 62°

C.

44. The method of claim 37, wherein each single-stranded oligonucleotide in said second plurality of single-stranded oligonucleotides is formed by joining an adjacent pair of oligonucleotides in said first plurality of single-stranded oligonucleotides.

45. The method of claim 37, wherein each single-stranded oligonucleotide in said fourth plurality of single-stranded oligonucleotides is formed by joining an adjacent pair of oligonucleotides in said third plurality of single-stranded oligonucleotides.

46. The method of claim 37, the method further comprising:

f) assembling said set of oligonucleotides by a polymerase chain reaction or a ligase chain reaction with an annealing temperature that is a predetermined amount lower than the lowest annealing temperature of said first predetermined annealing temperature range, thereby forming an assembly mixture that comprises said polynucleotide.

47. The method of claim 46, wherein the predetermined amount is 1°

C. or larger.

48. The method of claim 46, the method further comprising

g) cloning the polynucleotide into a vector.

49. The method of claim 46, wherein the assembly mixture comprises a plurality of different polynucleotide molecules, the method further comprising:

g) creating a plurality of heteroduplexes between different individual polynucleotide molecules within the plurality of different polynucleotide molecules in the assembly;

h) treating said plurality of heteroduplexes with an agent that binds preferentially to mismatched sequences within a double-stranded DNA molecule; and

i) using said agent to remove double-stranded DNA molecules containing mismatched sequences from the assembly mixture.

50. The method of claim 49, the method further comprising:

j) amplifying said polynucleotide by the polymerase chain reaction.

51. The method of claim 50, the method further comprising

k) cloning the polynucleotide into a vector.

52. The method of claim 37, wherein said at least one assembly criterion comprises a requirement that the annealing temperature of each intended complementary pair of single-stranded oligonucleotides in said set of oligonucleotides falls within a third predetermined temperature range.

53. The method of claim 52, wherein the third predetermined temperature range encompasses a total of 4°

C. or less.

54. The method of claim 52, wherein the third predetermined temperature range encompasses a total of 3°

C. or less.

55. The method of claim 37, wherein said at least one assembly criterion comprises a requirement that the single-stranded oligonucleotide length of each oligonucleotide in said set of oligonucleotides is within a predetermined oligonucleotide length range.

56. The method of claim 55, wherein the predetermined oligonucleotide length range is between 20 nucleotides and 70 nucleotides.

57. The method of claim 55, wherein the predetermined oligonucleotide length range is between 25 nucleotides and 65 nucleotides.

58. The method of claim 37, wherein said at least one assembly criterion comprises a requirement that the number of single-stranded oligonucleotides in the second plurality of single-stranded oligonucleotides matches the number of single-stranded oligonucleotides in the fourth plurality of single-stranded oligonucleotides.

59. The method of claim 37, wherein said at least one assembly criterion comprises a requirement that the annealing temperature of each pair of single-stranded oligonucleotides in said set of oligonucleotides for assembly, whose annealing is not intended for said assembly, is below a predetermined temperature.

60. The method of claim 59, wherein the predetermined temperature is the annealing temperature of a pair of oligonucleotides in the set of oligonucleotides whose annealing is intended for assembly of said polynucleotide.

61. The method of claim 59, wherein the predetermined temperature is at least 10°

C. below the annealing temperature of a pair of oligonucleotides in the set of oligonucleotides whose annealing is intended for assembly of said polynucleotide.

62. The method of claim 59, wherein the predetermined temperature is at least 15°

C. below the annealing temperature of a pair of oligonucleotides in the set of oligonucleotides whose annealing is intended for assembly of said polynucleotide.

63. The method of claim 59, wherein the predetermined temperature is at least 20°

C. below the annealing temperature of a pair of oligonucleotides in the set of oligonucleotides whose annealing is intended for assembly of said polynucleotide.

64. The method of claim 37, wherein said at least one assembly criterion comprises a requirement that a maximum length of a sequence that occurs more than once within the first strand of said polynucleotide and that is found at a terminus of any oligonucleotide in said set of oligonucleotides is less than a predetermined length.

65. The method of claim 64, wherein the predetermined length is 10 nucleotides or greater.

66. The method of claim 64, wherein the predetermined length is 12 nucleotides or greater.

67. The method of claim 37, wherein a pair of oligonucleotides in said set of oligonucleotides that are intended to be annealed to form said polynucleotide are not completely overlapping.

68. The method of claim 37, wherein a first single-stranded oligonucleotide has an n-mer overhang relative to a second single-stranded oligonucleotide in said set of oligonucleotides, and wherein annealing of said first single-stranded oligonucleotide and said second oligonucleotide single-stranded oligonucleotide is intended for assembly of said polynucleotide, where n is between 1 and 40.

69. The method of claim 37, wherein said at least one assembly criterion comprises a requirement that a predetermined length of a nucleotide sequence at a terminus of an oligonucleotide in said set of oligonucleotides is not found at either terminus of any other oligonucleotide in said set of oligonucleotides.

70. The method of claim 69, wherein said predetermined length is 5 nucleotides.

71. The method of claim 69, wherein said predetermined length is 4 nucleotides.

72. The method of claim 69, wherein said predetermined length is 3 nucleotides.

73. The method of claim 37, wherein said polynucleotide encodes a polypeptide, the method further comprising:

(i) selecting, prior to said identifying step a), an initial polynucleotide sequence for said polynucleotide that codes for said polypeptide, wherein a codon frequency in said initial polynucleotide sequence is determined by a codon bias table; and

(ii) modifying, prior to said identifying step a), an initial codon choice in said initial polynucleotide sequence for said polynucleotide in accordance with a design criterion, thereby constructing a final polynucleotide sequence for said polynucleotide that codes for said polypeptide.

74. The method of claim 73, wherein the design criterion comprises one or more of:

(i) exclusion of a restriction site sequence in said initial polynucleotide sequence;

(ii) incorporation of a restriction site sequence in said initial polynucleotide sequence;

(iii) a designation of a target G+C content in the initial polynucleotide sequence;

(iv) an allowable length of a sub-sequence that can be exactly repeated within either strand of the initial polynucleotide sequence;

(v) an allowable annealing temperature of any sub-sequence to any other sub-sequence within either strand of the initial polynucleotide sequence;

(vi) exclusion of a hairpin turn in the initial polynucleotide sequence;

(vii) exclusion of a repeat element in the initial polynucleotide sequence;

(viii) exclusion of a ribosome binding site in the initial polynucleotide sequence;

(ix) exclusion of a polyadenylation signal in the initial polynucleotide sequence;

(x) exclusion of a splice site in the initial polynucleotide sequence;

(xi) exclusion of an open reading frame in each possible 5′

reading frame in the initial polynucleotide sequence;

(xii) exclusion of a polynucleotide sequence that facilitates RNA degradation in the initial polynucleotide sequence;

(xiii) exclusion of an RNA polymerase termination signal in the initial polynucleotide sequence;

(xiv) exclusion of a transcriptional promoter in the initial polynucleotide sequence;

(xv) exclusion of an immunostimulatory sequence in the initial polynucleotide sequence;

(xvi) incorporation of an immunostimulatory sequence in the initial polynucleotide sequence;

(xvii) exclusion of an RNA methylation signal in the initial polynucleotide sequence;

(xviii) exclusion of a selenocysteine incorporation signal in the initial polynucleotide sequence;

(xix) exclusion of an RNA editing sequence in the initial polynucleotide sequence;

(xx) exclusion of an RNAi-targeted sequence in the initial polynucleotide sequence; and

(xxi) exclusion of an inverted repeat within the first 45 nucleotides encoding said synthetic polypeptide in the initial polynucleotide sequence.

75. The method of claim 73, wherein the design criterion comprises:

reduced sequence identity to a reference polynucleotide, and wherein

modifying said initial codon choice in said initial polynucleotide in accordance with said design criterion comprises altering a codon choice in said initial polynucleotide sequence to reduce sequence identity to said reference polynucleotide.

76. The method of claim 73, wherein the design criterion comprises:

increased sequence identity to a reference polynucleotide, and wherein

modifying said initial codon choice in said initial polynucleotide in accordance with said design criterion comprises altering a codon choice in said initial polynucleotide sequence to increase sequence identity to said reference polynucleotide.

77. A computer program product for use in conjunction with a computer system, the computer program product comprising a computer readable storage medium and a computer program mechanism embedded therein, the computer program mechanism comprising instructions for performing the method of claim 37.

82. A computer system comprising:

a central processing unit;

a memory, coupled to the central processing unit, the memory storing instructions for carrying out the method of claim 37.

78-81. -81. (canceled)

83-86. -86. (canceled)

87. A method of designing a single designed polynucleotide having a sequence, the method comprising:

a) dividing the sequence into a plurality of polynucleotide sub-sequences;

b) adding a first restriction site to a 3′

end of a first polynucleotide sub-sequence in said plurality of polynucleotide sub-sequences;

c) adding a second restriction site to a 5′

end of a second polynucleotide sub-sequence in said plurality of polynucleotide sub-sequences, such that cleavage of said first restriction site and said second restriction site would cause a terminal portion of said first polynucleotide sub-sequence to become complementary with a terminal portion of said second polynucleotide sub-sequence;

d) for each respective sub-sequence in said plurality of polynucleotide sub-sequences;

(i) identifying a first plurality of single-stranded oligonucleotides that collectively encode all or a portion of a first strand of said respective sub-sequence, wherein each respective single-stranded oligonucleotide in said first plurality of single-stranded oligonucleotides is characterized by an annealing temperature to its exact complement that is in a first predetermined annealing temperature range;

(ii) identifying a second plurality of single-stranded oligonucleotides from said first plurality of single-stranded oligonucleotides, wherein a single-stranded oligonucleotide in said second plurality of single-stranded oligonucleotides is formed by joining an adjacent pair of oligonucleotides in said first plurality of single-stranded oligonucleotides;

(iii) identifying a third plurality of single-stranded oligonucleotides that collectively encode all or a portion of a second strand of said respective sub-sequence, wherein each respective single-stranded oligonucleotide in said third plurality of single-stranded oligonucleotides is characterized by an annealing temperature to its exact complement that is in a second predetermined annealing temperature range;

(iv) identifying a fourth plurality of single-stranded oligonucleotides from said third plurality of single-stranded oligonucleotides, wherein a single-stranded oligonucleotide in said fourth plurality of single-stranded oligonucleotides is formed by joining an adjacent pair of oligonucleotides in said third plurality of single-stranded oligonucleotides;

wherein a set of oligonucleotides comprises said second plurality of oligonucleotides and said fourth plurality of oligonucleotides; and

(v) determining whether the set of oligonucleotides satisfies at least one assembly criterion, wherein

when the set of oligonucleotides satisfies said at least one assembly criterion, the set of oligonucleotides is selected; and

when the set of oligonucleotides does not satisfy said at least one assembly criterion, the set of oligonucleotides is rejected and steps (i) through (v) are repeated.

e) repeating steps a) through d) when a set of oligonucleotides has not been selected for each respective sub-sequence in said plurality of polynucleotide sub-sequences.

88. The method of claim 87, wherein the first restriction site is for a restriction enzyme that cleaves outside its recognition sequence.

89. The method of claim 87, wherein the first restriction site is a type IIs site.

90. The method of claim 87, wherein the second restriction site is for a restriction enzyme that cleaves outside its recognition sequence.

91. The method of claim 87, wherein the second restriction site is a type IIs site.

92. The method of claim 87, the method further comprising:

f) assembling the set of oligonucleotides for a respective sub-sequence in said plurality of polynucleotide subsequences using a polymerase chain reaction or a ligase chain reaction with an annealing temperature that is a predetermined amount lower than the lowest annealing temperature of any intended complementary pair of single-stranded oligonucleotides in said set of oligonucleotides; and

g) repeating step f) for each respective sub-sequence in said plurality of polynucleotide sub-sequences.

93. The method of claim 92, the method further comprising:

h) cloning each polynucleotide sub-sequence into a vector; and

i) obtaining each cloned polynucleotide sub-sequence from its vector and joining the sub-sequences to form the single designed polynucleotide.

94. A computer program product for use in conjunction with a computer system, the computer program product comprising a computer readable storage medium and a computer program mechanism embedded therein, the computer program mechanism for designing a single designed polynucleotide having a sequence, the computer program mechanism comprising instructions for carrying out the method of claim 87.

95. A computer system for designing a single designed polynucleotide having a sequence, the computer system comprising instructions for carrying out the method of claim 87.

96-155. -155. (canceled)