Incrementally Truncated Nucleic Acids and Methods of Making Same

• US 20080227659A1
• Filed: 02/19/2008
• Published: 09/18/2008
• Est. Priority Date: 11/15/2000
• Status: Active Grant

• Current Assignee: Andrew E. Nixon, Marc Ostermeier, Stefan Lutz, Stephen J. Benkovic
• Original Assignee: Andrew E. Nixon, Marc Ostermeier, Stefan Lutz, Stephen J. Benkovic
• Inventors: Benkovic, Stephen J., Lutz, Stefan, Nixon, Andrew E., Ostermeier, Marc
• Granted Patent: US 7,820,413 B2
• Time in Patent Office: Days
• US Class Current: 506/16
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms

Abstract

A series of methods that utilize the incremental truncation of nucleic acids are described to create a plurality of modified nucleic acids and hybrid polypeptides. A plurality of substantially all possible single base-pair deletions of a given nucleic acid sequence is created. A method of making shuffled incremental truncated nucleic acids, which is independent of nucleic acid sequence homology, is also described. These methods can be used in protein engineering, protein folding, protein evolution, and the chemical synthesis of novel hybrid proteins and polypeptides.

Claims

1. A method of making a plurality of expression products of an incrementally truncated nucleic acid comprising the steps of:

a) providing a parent nucleic acid;

b) serially removing nucleotides from one or both termini of said nucleic acid to form truncated parent nucleic acids whose length decreases incrementally over time;

c) stopping said serial nucleotide removal at a plurality of different times to form a plurality of incrementally truncated nucleic acids; and

d) expressing said plurality of incrementally truncated nucleic acids to form a plurality of truncated nucleic acid expression products.

2. An incrementally truncated nucleic acid made by the process of claim 1.

3. A truncated nucleic acid expression product made by the process of claim 1.

4. The method of claim 1 wherein said parent nucleic acid comprises a library of nucleic acids.

5. A method of making a plurality of incrementally truncated hybrid nucleic acids comprising the steps of:

a) providing a first and second parent nucleic acid;

b) serially removing nucleotides from one or both termini of said first and second parent nucleic acids to form truncated first and second parent nucleic acids whose length decreases incrementally over time;

c) stopping said serial nucleotide removal at a plurality of different times to form a plurality of incrementally truncated first and second parent nucleic acids; and

d) linking separate incrementally truncated first parent nucleic acids to separate incrementally truncated second parent nucleic acids to form a plurality of incrementally truncated hybrid nucleic acids.

6. A method of making a plurality of transformed incrementally truncated hybrid nucleic acids comprising the step of transforming said plurality of incrementally truncated hybrid nucleic acids of claim 5 into a plurality of hosts to form a plurality of transformed incrementally truncated hybrid nucleic acids.

7. An incrementally truncated hybrid nucleic acid made by the process of claim 5.

8. A transformed incrementally truncated hybrid nucleic acid made by the process of claim 6.

9. The method of claim 5 wherein said parent nucleic acids comprise a library of nucleic acids.

10. A method of making a plurality of first variant incrementally truncated hybrid nucleic acids comprising the steps of:

a) providing a first and second parent nucleic acid;

b) serially removing nucleotides from one or both termini of said first and second parent nucleic acids to form truncated first and second parent nucleic acids whose length decreases incrementally over time;

c) stopping said serial nucleotide removal at a plurality of different times to form a plurality of incrementally truncated first and second parent nucleic acids;

d) linking separate incrementally truncated first parent nucleic acids to separate incrementally truncated second parent nucleic acids to form a plurality of first variant incrementally truncated hybrid nucleic acids, wherein said incrementally truncated first parent nucleic acids form the N-terminal coding sequence of each of said first variant incrementally truncated hybrid genes.

11. A method of making a plurality of transformed first variant incrementally truncated hybrid nucleic acids comprising the step of transforming the first variant incrementally truncated hybrid nucleic acids of claim 10 into a plurality of hosts to form a plurality of transformed first variant incrementally truncated hybrid nucleic acids.

12. A first variant incrementally truncated hybrid nucleic acid made according to the method of claim 10.

13. A transformed first variant incrementally truncated hybrid nucleic acid made according to the method of claim 11.

14. The method of claim 10 wherein said parent nucleic acids comprise a library of nucleic acids.

15. A method of making a plurality of second variant incrementally truncated hybrid nucleic acids comprising the steps of:

a) providing a first and second parent nucleic acid;

b) serially removing nucleotides from one or both termini of said first and second parent nucleic acids to form truncated first and second parent nucleic acids whose length decreases incrementally over time;

c) stopping said serial nucleotide removal at a plurality of different times to form a plurality of incrementally truncated first and second parent nucleic acids;

d) linking separate incrementally truncated first parent nucleic acids to separate incrementally truncated second parent nucleic acids to form a plurality of second variant incrementally truncated hybrid nucleic acids, wherein said incrementally truncated second parent nucleic acids form the N-terminal coding sequence of each of said second variant incrementally truncated hybrid genes.

16. A method of making a plurality of transformed second variant incrementally truncated hybrid nucleic acids comprising the step of transforming the second variant incrementally truncated hybrid nucleic acids of claim 15 into a plurality of hosts to form a plurality of transformed second variant incrementally truncated hybrid nucleic acids.

17. A second variant incrementally truncated hybrid nucleic acid made according to the method of claim 15.

18. A transformed second variant incrementally truncated hybrid nucleic acid made according to the method of claim 16.

19. The method of claim 15 wherein said parent nucleic acids comprise a library of nucleic acids.

20. A method of making a plurality of shuffled incrementally truncated nucleic acids comprising the steps of:

a) providing isolated nucleic acid inserts from a plurality of incremental truncation modified nucleic acids;

b) recombining said isolated nucleic acid inserts for a time period and under conditions suitable to form a plurality of shuffled incrementally truncated hybrid nucleic acids.

21. A method of making a plurality of transformed shuffled incrementally truncated nucleic acids comprising the step of transforming the plurality of shuffled incrementally truncated nucleic acids of claim 20 into a plurality of hosts to make a plurality of transformed shuffled incrementally truncated nucleic acids.

22. A shuffled incrementally truncated nucleic acid made according to the process of claim 20.

23. A transformed shuffled incrementally truncated nucleic acid made according to the process of claim 21.

24. A method of making a plurality of analog-containing incrementally truncated nucleic acids comprising the steps of:

a) providing a plurality of nucleotide analog-containing parent nucleic acids; and

b) removing nucleotides from said plurality of nucleotide analog-containing parent nucleic acids with a nuclease enzyme that does not depolymerize nucleotide analogs incorporated into a nucleic acid under conditions and for a time period sufficient to form a plurality of analog-containing incrementally truncated nucleic acids.

25. A method of making a plurality of transformed analog-containing incrementally truncated nucleic acids comprising the step of transforming the plurality of analog-containing incrementally truncated nucleic acids of claim 24 into a plurality of hosts to form a plurality of transformed analog-containing incrementally truncated nucleic acids.

26. The method of claim 24 wherein said plurality of nucleotide analog-containing parent nucleic acids comprises a plurality of nucleotide analog-containing incremental truncation modified nucleic acids.

27. The method of claim 26 wherein said plurality of nucleotide analog-containing incremental truncation modified nucleic acids comprises a plurality of nucleotide analog-containing shuffled incrementally truncated hybrid nucleic acids.

28. The method of claim 24 wherein said nuclease enzyme that does not depolymerize nucleotide analogs incorporated into a nucleic acid is exonuclease III.

29. A method of creating a plurality of circular permutation incremental truncation hybrid nucleic acids comprising the steps of:

a) providing first and second nucleic acids;

b) inserting a plurality of circularly permuted nucleic acid fragments containing a randomly located restriction enzyme site between said first and second nucleic acids to form a plurality of circular permutation hybrids;

c) reacting said plurality of circular permutation hybrids with a restriction enzyme that recognizes and specifically hydrolyzes said randomly located restriction enzyme site to form a plurality of circular permutation incremental truncation substrates;

d) removing nucleotides from both ends of said restriction enzyme site to form a plurality of circular permutation incrementally truncated hybrid nucleic acids;

e) stopping said serial nucleotide removal to form a plurality of circular permutation incrementally truncated hybrid nucleic acids having a gap; and

f) closing said gap to form a plurality of circular permutation incremental truncation hybrid nucleic acids.

30. A method of making a plurality of transformed circular permutation incremental truncation hybrid nucleic acids comprising the step of transforming said plurality of circular permutation incremental truncation hybrid nucleic acids of claim 29 into a plurality of hosts to form a plurality of transformed circular permutation incremental truncation hybrid nucleic acids.

31. A plurality of truncated nucleic acid expression products.

32. A plurality of incrementally truncated hybrid nucleic acids.

33. A plurality of first variant incrementally truncated hybrid nucleic acids.

34. A plurality of second variant incrementally truncated hybrid nucleic acids.

35. A plurality of shuffled incrementally truncated hybrid nucleic acids.

36. A plurality of nucleotide analog-containing incrementally truncated nucleic acids.

37. A plurality of circular permutation incremental truncation hybrid nucleic acids.