Methods and apparatus for synthesizing nucleic acids
The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using enzymes and specially designed nucleotide analogs. Using the methods of the invention, specific sequences of polynucleotides can be synthesized de novo, base by base, in an aqueous environment, without the use of a nucleic acid template. Because the nucleotide analogs have an unmodified 3′ OH, i.e., as found in “natural” deoxyribose and ribose molecules, the analogs result in natural polynucleotides suitable for incorporation into biological systems.
1. A method for synthesizing an oligonucleotide, comprising:
exposing an oligonucleotide attached to a solid support to a nucleotide analog in the presence of a nucleotidyl transferase enzyme and in the absence of a nucleic acid template such that the nucleotide analog is incorporated into the oligonucleotide,
wherein the nucleotide analog comprises a nucleotide coupled, by a cleavable linker, to an inhibitor comprising a negatively-charged moiety and an amino acid with a negative charge that prevents the nucleotidyl transferase from catalyzing incorporation of a nucleotide or an additional nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker.
2. The method of claim 1 , wherein the nucleotide analog comprises the following structure:
3. The method of claim 1 , wherein the nucleotide analog comprises the following structure:
4. The method of claim 1 , wherein the nucleotide analog comprises the following cleavable linker structure:
wherein NTP=nucleotide triphosphate, n=1, 2, 3, or 4, and —X— is —O—, —S—, —NH—, or —CH 2 —.
5. The method of claim 1 , wherein the nucleotide analog has the following cleavable linker structure:
wherein NTP=nucleotide triphosphate, —X— is —O—, —S—, —NH—, or —CH 2 —.
6. The method of claim 1 , wherein the nucleotide analog has the following inhibitor structure:
wherein NTP=nucleotide triphosphate, n=1, 2, 3, or 4, and —X— is —O—, —S—, —NH—, or —CH 2 —.
7. The method of claim 1 , wherein the nucleic acid attached to the solid support is exposed to the nucleotide analog in the presence of an aqueous solution having a pH between about 6.5 and 8.5.
8. The method of claim 1 , the nucleic acid attached to the solid support is exposed to the nucleotide analog in the presence of an aqueous solution at a temperature between about 35 and 39° C.
9. The method of claim 1 , wherein the solid support is a bead, a well, or a peg.
10. The method of claim 1 , wherein the nucleic acid is single stranded.
11. The method of claim 1 , wherein the cleavable linker comprises a moiety that forms a cyclic by-product when cleaved from the nucleotide analog.
12. The method of claim 1 , further comprising:
cleaving the cleavable linker in order to produce a native nucleotide; and
exposing the native nucleotide to a second nucleotide analog in the presence of a nucleotidyl transferase enzyme and in the absence of a nucleic acid template.
13. The method of claim 1 , further comprising providing an aqueous solution comprising the nucleotide analog and the nucleotidyl transferase enzyme.
14. The method of claim 1 , wherein the amino acid is acetylated.
15. The method of claim 1 , wherein the nucleotide analog comprises a ribose sugar or a deoxyribose sugar.
16. The method of claim 1 , wherein the nucleotide substrate comprises a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil.
17. The method of claim 1 , wherein the nucleotide analog is encapsulated in a droplet.
18. The method of claim 1 , wherein the nucleotidyl transferase enzyme is encapsulated in a droplet.
19. A method for synthesizing an oligonucleotide, comprising:
exposing an oligonucleotide attached to a solid support to a nucleotide analog in the presence of a nucleotidyl transferase enzyme and in the absence of a nucleic acid template such that the nucleotide analog is incorporated into the oligonucleotide,
wherein the nucleotide analog comprises a nucleotide coupled, by a cleavable linker, to an inhibitor comprising a positively-charged moiety and an amino acid with a positive charge that prevents the nucleotidyl transferase from catalyzing incorporation of a nucleotide or an additional nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker.
20. The method of claim 19 , wherein the nucleotide analog comprises the following structure:
21. The method of claim 19 , wherein the nucleotide analog comprises the following structure:
22. The method of claim 19 , wherein the nucleotide analog comprises the following cleavable linker structure:
wherein NTP=nucleotide triphosphate, n=1, 2, 3, or 4, and —X— is —O—, —S—, —NH—, or —CH 2 —.
23. The method of claim 19 , wherein the nucleotide analog has the following cleavable linker structure:
wherein NTP=nucleotide triphosphate, —X— is —O—, —S—, —NH—, or —CH 2 —.
24. The method of claim 19 , wherein the nucleotide analog has the following inhibitor structure:
wherein NTP=nucleotide triphosphate, n=1, 2, 3, or 4, and —X— is —O—, —S—, —NH—, or —CH 2 —.
25. The method of claim 19 , wherein the nucleic acid attached to the solid support is exposed to the nucleotide analog in the presence of an aqueous solution having a pH between about 6.5 and 8.5.
26. The method of claim 19 , the nucleic acid attached to the solid support is exposed to the nucleotide analog in the presence of an aqueous solution at a temperature between about 35 and 39° C.
27. The method of claim 19 , wherein the solid support is a bead, a well, or a peg.
28. The method of claim 19 , wherein the nucleic acid is single stranded.
29. The method of claim 19 , wherein the cleavable linker comprises a moiety that forms a cyclic by-product when cleaved from the nucleotide analog.
30. The method of claim 19 , further comprising:
cleaving the cleavable linker in order to produce a native nucleotide; and
exposing the native nucleotide to a second nucleotide analog in the presence of a nucleotidyl transferase enzyme and in the absence of a nucleic acid template.
31. The method of claim 19 , further comprising providing an aqueous solution comprising the nucleotide analog and the nucleotidyl transferase enzyme.
32. The method of claim 19 , wherein the amino acid is acetylated.
33. The method of claim 19 , wherein the nucleotide analog comprises a ribose sugar or a deoxyribose sugar.
34. The method of claim 19 , wherein the nucleotide substrate comprises a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil.
35. The method of claim 19 , wherein the nucleotide analog is encapsulated in a droplet.
36. The method of claim 19 , wherein the nucleotidyl transferase enzyme is encapsulated in a droplet.
37. A method for synthesizing an oligonucleotide, comprising:
exposing an oligonucleotide attached to a solid support to a nucleotide analog in the presence of a nucleotidyl transferase enzyme and in the absence of a nucleic acid template such that the nucleotide analog is incorporated into the oligonucleotide,
wherein the nucleotide analog has the following inhibitor structure:
wherein NTP=nucleotide triphosphate, n=1, 2, 3, or 4, and —X— is —O—, —S—, —NH—, or —CH 2 —.
38. The method of claim 37 , wherein the nucleic acid attached to the solid support is exposed to the nucleotide analog in the presence of an aqueous solution having a pH between about 6.5 and 8.5.
39. The method of claim 37 , the nucleic acid attached to the solid support is exposed to the nucleotide analog in the presence of an aqueous solution at a temperature between about 35 and 39° C.
40. The method of claim 37 , wherein the solid support is a bead, a well, or a peg.
41. The method of claim 37 , wherein the nucleic acid is single stranded.
42. The method of claim 37 , further comprising:
cleaving the cleavable linker in order to produce a native nucleotide; and
exposing the native nucleotide to a second nucleotide analog in the presence of a nucleotidyl transferase enzyme and in the absence of a nucleic acid template.
43. The method of claim 37 , further comprising providing an aqueous solution comprising the nucleotide analog and the nucleotidyl transferase enzyme.
44. The method of claim 37 , wherein the nucleotide analog comprises a ribose sugar or a deoxyribose sugar.
45. The method of claim 37 , wherein the nucleotide substrate comprises a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil.
46. The method of claim 37 , wherein the nucleotide analog is encapsulated in a droplet.
47. The method of claim 37 , wherein the nucleotidyl transferase enzyme is encapsulated in a droplet.