Methods for making character strings, polynucleotides and polypeptides having desired characteristics

• US 7,024,312 B1
• Filed: 07/18/2000
• Issued: 04/04/2006
• Est. Priority Date: 01/19/1999
• Status: Expired due to Term

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: del Cardayre, Stephen, Tobin, Matthew, Patten, Phillip A., Giver, Lorraine J., Selifonov, Sergey A., Gustafsson, Claes, Minshull, Jeremy, Stemmer, Willem P. C.
• Primary Examiner(s): Brusca, John
• Assistant Examiner(s): Zhou, Shubo (Joe)
• Application Number:
  US09/618,579
• Time in Patent Office: 2,086 Days
• Field of Search: 435/6, 435/15, 435/91.2, 435/91.1, 435/192, 435/197, 435/4, 536/23.1, 536/24.1, 530/300, 530/350, 702/19, 702/20
• US Class Current: 702/19
• CPC Class Codes: B01J 2219/007 Simulation or vitual synthesis
• CPC Class Codes: G16B 10/00 ICT specially adapted for evolutionary bioinformatics, e.g. phylogenetic tree construction or analysis
• CPC Class Codes: G16B 15/00 ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
• CPC Class Codes: G16B 15/20 Protein or domain folding
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 20/30 Detection of binding sites or motifs
• CPC Class Codes: G16B 20/50 Mutagenesis
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search
• CPC Class Codes: G16B 30/20 Sequence assembly
• CPC Class Codes: G16B 35/00 ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
• CPC Class Codes: G16B 35/20 Screening of libraries
• CPC Class Codes: G16B 40/00 ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
• CPC Class Codes: G16B 40/10 Signal processing, e.g. from mass spectrometry [MS] or from PCR
• CPC Class Codes: G16C 20/60 In silico combinatorial chemistry

Abstract

“In silico” nucleic acid recombination methods, related integrated systems utilizing genetic operators and libraries made by in silico shuffling methods are provided.

Claims

1. A method of identifying a set of oligonucleotides for use in an in vitro recombination procedure, the method comprising:

(a) providing data identifying sequences of two or more parental polypeptides or parental nucleic acids that encode the polypeptides;

(b) computationally selecting one or more cross-over sites on the sequences based on structural information about the parental polypeptides or polypeptides encoded by the parental nucleic acids;

thereby defining one or more recombinant polypeptides or recombinant nucleic acids that result from cross-overs between the parental polypeptides or nucleic acids at the one or more cross-over sites;

(c) selecting at least one of the recombinant polypeptides or recombinant nucleic acids by computationally assessing structural stability of at least some of the recombinant polypeptides or polypeptides encoded by the recombinant nucleic acids; and

(d) computationally identifying one or more oligonucleotides for in vitro recombination by choosing at least one portion of at least one of the recombinant polypeptides or recombinant nucleic acids selected in (c) which at least one portion corresponds to the one or more oligonucleotides identified for in vitro recombination.

2. The method of claim 1, wherein the structural information employed in (b) comprises information depicting the three-dimensional structure of at least a portion of the parental polypeptides or polypeptides encoded by the parental nucleic acids.

3. The method of claim 1, wherein (b) comprises selecting cross-over points that correspond to overlapping amino acids in the parental polypeptides.

4. The method of claim 1, wherein (b) comprises selecting cross-over points at sites that will preserve selected subunits, domains, or motifs in the parental polypeptides.

5. The method of claim 1, wherein (b) comprises selecting cross-over points at sites chosen to maintain or disrupt one ore more structural relationships between two or more amino acids in the parental polypeptides.

6. The method of claim 1, further comprising performing an additional genetic operation on one or more of the parental or recombinant polypeptides or the parental or recombinant nucleic acids.

7. The method of claim 1, wherein the genetic operation is selected from the group consisting of multiplication, mutation, fragmentation, and ligation.

8. The method of claim 1, wherein the two or more parental polypeptides or parental nucleic acids comprise naturally occurring polypeptides or naturally occurring nucleic acids that encodes a polypeptides.

9. The method of claim 1, wherein (c) comprises computationally assessing three-dimensional structural stability of at least some of the recombinant polypeptides or polypeptides encoded by the recombinant nucleic acids.

10. The method of claim 1, wherein the oligonucleotides identified in (d) are “

bridging”

oligonucleotides.

11. The method of claim 1, further comprising aligning the two or more parental polypeptides or parental nucleic acids prior to computationally selecting one or more cross-over sites on the sequences.

12. The method of claim 1, wherein the recombinant polypeptides or recombinant nucleic acids that result from the cross-overs comprise contiguous sequences of multiple amino acids or nucleotides from the two or more of the parental polypeptides or parental nucleic acids.

13. A computer program product comprising a machine readable medium on which is provided program instructions for identifying a set of oligonucleotides for use in an in vitro recombination procedure, the program instructions comprising:

(a) code for providing data identifying sequences of two or more parental polypeptides or parental nucleic acids that encode the polypeptides;

(b) code for selecting one or more cross-over sites on the sequences based on structural information about the parental polypeptides or polypeptides encoded by the parental nucleic acids;

thereby defining one or more recombinant polypeptides or recombinant nucleic acids that result from cross-overs between the parental polypeptides or nucleic acids at the one or more cross-over sites;

(c) code for selecting at least one of the recombinant polypeptides or recombinant nucleic acids by assessing structural stability of at least some of the recombinant polypeptides or polypeptides encoded by the recombinant nucleic acids; and

(d) code for identifying one or more oligonucleotides for in vitro recombination by choosing at least one portion of at least one of the recombinant polypeptides or recombinant nucleic acids selected in (c) which at least one portion corresponds to the one or more oligonucleotides identified for in vitro recombination.

14. The computer program product of claim 13, wherein the structural information employed in (b) comprises information depicting the three-dimensional structure of at least a portion of the parental polypeptides or polypeptides encoded by the parental nucleic acids.

15. The computer program product of claim 13, wherein (b) comprises code for selecting cross-over points that correspond to overlapping amino acids in the parental polypeptides.

16. The computer program product of claim 13, wherein (b) comprises code for selecting cross-over points at sites that will preserve selected subunits, domains, or motifs in the parental polypeptides.

17. The computer program product of claim 13, wherein (b) comprises code for selecting cross-over points at sites chosen to maintain or disrupt one or more structural relationships between two or more amino acids in the parental polypeptides.

18. The computer program of claim 13, further comprising code for performing an additional genetic operation on one or more of the parental or recombinant polypeptides or the parental or recombinant nucleic acids.

19. The computer program product of claim 18, wherein the genetic operation is selected from the group consisting of multiplication, mutation, fragmentation, and ligation.

20. The computer program product of claim 13, wherein at least one of the parental polypeptides or the parental nucleic acids comprise a naturally occurring polypeptide or a naturally occurring nucleic acid that encodes a polypeptide.

21. The computer program product of claim 13, wherein (c) comprises code for assessing three-dimensional structural stability of at least some of the recombinant polypeptides or polypeptides encoded by the recombinant nucleic acids.

22. The computer program product of claim 13, wherein the code for identifying one or more oligonucleotides in (d) identifies “

bridging”

oligonucleotides.

23. The computer program product of claim 13, further comprising code for aligning the two or more parental polypeptides or parental nucleic acids prior to computationally selecting one or more cross-over sites on the sequences.

24. The computer program product of claim 13, wherein the recombinant polypeptides or recombinant nucleic acids that result from the cross-overs comprise contiguous sequences of multiple amino acids or nucleotides from the two or more of the parental polypeptides or parental nucleic acids.