Method for making polynucleotides having desired characteristics

• US 7,853,410 B2
• Filed: 10/18/2007
• Issued: 12/14/2010
• Est. Priority Date: 01/19/1999
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, 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): Zhou; Shubo (Joe)
• Application Number:
  US11/975,638
• Publication Number:
  US 20080050782A1
• Time in Patent Office: 1,153 Days
• Field of Search: None
• 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 making a recombinant nucleic acid, the method comprising:

providing a plurality of parental character strings corresponding to a plurality of nucleic acids, which character strings, when aligned for maximum identity, comprise at least one region of heterology;

aligning the character strings;

defining a set of character string subsequences, which set of subsequences comprises subsequences of at least two of the plurality of parental character strings;

providing a set of oligonucleotides corresponding to the set of character string subsequences;

annealing members of the set of oligonucleotides with one another in vitro;

after annealing, elongating one or more members of the set of oligonucleotides with a polymerase, or ligating at least two members of the set of oligonucleotides with a ligase, thereby producing one or more recombinant nucleic acid;

selecting the recombinant nucleic acid, or a polypeptide encoded by the recombinant nucleic acid for a desired trait or property, thereby providing a selected recombinant nucleic acid; and

recombining the selected recombinant nucleic acid with one or more of (i) a nucleic acid homologous to a parental character string, and (ii) an oligonucleotide member from the set of oligonucleotides.

2. The method of claim 1, wherein each parental character string when aligned for maximum identity contains areas of identity and areas of heterology with any other parental character string.

3. The method of claim 1, wherein at least one of the parental character strings is an evolutionary or artificial intermediate.

4. The method of claim 1, wherein at least one of the parental character strings corresponds to a designed nucleic acid or designed polypeptide.

5. The method of claim 4, wherein the designed nucleic acid represents an energy minimized design for an encoded polypeptide.

6. The method of claim 1, further comprising applying one or more genetic operator to one or more of the parental character strings, or to one or more of the character string subsequences, wherein the genetic operator is selected from the group consisting of:

a mutation of the one or more parental character strings or one or more character string subsequences, a multiplication of the one or more parental character strings or one or more character string subsequences, a fragmentation of the one or more parental character strings or one or more character string subsequences, a crossover between any of the one or more parental character strings or one or more character string subsequences or an additional character string, a ligation of the one or more parental character strings or one or more character string subsequences, an elitism calculation, a calculation of sequence homology or sequence similarity of aligned strings, a recursive use of one or more genetic operator for evolution of character strings, application of a randomness operator to the one or more parental character strings or the one or more character string subsequences, a deletion mutation of the one or more parental character strings or one or more character string subsequences, an insertion mutation into the one or more parental character strings or one or more of character string subsequences, subtraction of the of the one or more parental character strings or one or more character string subsequences with an inactive sequence, selection of the of the one or more parental character strings or one or more character string subsequences with an active sequence, and death of the one or more parental character strings or one or more of character string subsequences.

7. The method of claim 1, further comprising generating a diplomat sequence, which diplomat sequence comprises an intermediate level of sequence similarity between two or more additional members of the plurality of parental character strings wherein the set of oligonucleotides comprises or encodes subsequences of the diplomat sequence.

8. The method of claim 1, further comprising selecting one or more cross-over sites between two or more of the parental character strings and wherein the set of oligonucleotides comprises one or more bridging oligonucleotides.

9. The method of claim 8, wherein the two or more parental sequences are less than 50% similar.

10. The method of claim 8, further comprising determining a sequence for one or more putative recombinant nucleic acids or polypeptides resulting from in silico recombination of the two or more parental character strings at the cross-over sites, and performing one or more in silico simulations of activity for one or more of the putative recombinant nucleic acid or polypeptide.

11. The method of claim 10, further comprising synthesizing the putative recombinant nucleic acid by providing fragments of the two or more parental nucleic acids and at least one of the corresponding bridge oligonucleotides, hybridizing the fragments and the bridge oligonucleotides and elongating the hybridized fragments with a polymerase or a ligase.

12. The method of claim 1, wherein the set of oligonucleotides comprise a plurality of overlapping oligonucleotides.

13. The method of claim 1, wherein the set of character string subsequences is defined by subdividing at least two of the plurality of parental character strings into segments of a selected length.

14. The method of claim 1, wherein aligning the character strings is performed in a digital computer or in a network-based system.

15. The method of claim 1, wherein providing the set of oligonucleotides comprises synthesizing a set of single-stranded oligonucleotides corresponding to the set of character string subsequences.

16. The method of claim 1, wherein the set of oligonucleotides is provided by synthesizing the oligonucleotides to comprise one or more modified parental character string subsequence, which subsequence comprises one or more of:

a parental character string subsequence modified by one or more replacement of one or more character of the parental character string subsequence with one or more different character;

a parental character string subsequence modified by one or more deletion or insertion of one or more characters of the parental character string subsequence;

a parental character string subsequence modified by inclusion of a degenerate sequence character at one or more randomly or non-randomly selected positions;

a parental character string subsequence modified by inclusion of a character string from a different character string from a second parental character string subsequence at one or more position;

a parental character string subsequence which is biased based upon its frequency in a selected library of nucleic acids; and

,

a parental character string subsequence which comprises, or encodes a polypeptide that comprises, one or more sequence motif, which sequence motif is artificially included in the subsequence.

17. The method of claim 16, wherein the sequence motif comprises an N-linked glycosylation sequence, an O-linked glycosylation sequence, a protease sensitive sequence, a collagenase sensitive sequence, a Rho-dependent transcriptional termination sequence, an RNA secondary structure sequence that affects the efficiency of transcription, an RNA secondary structure sequence that affects the efficiency of translation, a transcriptional enhancer sequence, a transcriptional promoter sequence, or a transcriptional silencing sequence.

18. The method of claim 1, wherein the oligonucleotide set contains one or more altered or degenerate positions as compared to the corresponding subsequence of one or more parental character string.

19. The method of claim 1, wherein selecting the recombinant nucleic acid is based upon its hybridization to a selected nucleic acid or to a set of selected nucleic acids.

20. The method of claim 1, wherein the character string subsequences from the at least two parental character strings are separated by a crossover point.

21. The method of claim 20, wherein the crossover point is selected by aligning at least one substring of each of at least two of the parental character strings to display pairwise identity between the substrings, and selecting a point within the aligned sequence as the crossover point.

22. The method of claim 20, wherein the crossover point is selected randomly.

23. The method of claim 20, wherein the crossover point is selected non randomly.

24. The method of claim 20, wherein the crossover point is selected non randomly by selecting a crossover point approximately in the middle of one or more identified pairwise identity region.

25. The method of claim 1, wherein providing the set of oligonucleotides comprises providing one or more oligonucleotide member of the set of oligonucleotides at a concentration which is higher than at least one or more additional oligonucleotide member of the set of oligonucleotides.

26. The method of claim 1, further comprising denaturing the selected recombinant nucleic acid, and contacting the recombinant nucleic acid with at least one additional nucleic acid from the oligonucleotide set.

27. The method of claim 1, further comprising denaturing the selected recombinant nucleic acid, and contacting the one or more recombinant nucleic acid with at least one additional nucleic acid produced by cleavage of at least one parental nucleic acid.

28. The method of claim 1, further comprising denaturing the selected recombinant nucleic acid, and contacting the recombinant nucleic acid with at least one additional nucleic acid produced by cleavage of a parental nucleic acid, which parental nucleic acid is cleaved by one or more of:

chemical cleavage, cleavage with a DNAse, and cleavage with a restriction endonuclease.

29. The method of claim 1, wherein at least one parental nucleic acid encodes a protein selected from:

erythropoietin (EPO), insulin, a peptide hormone, a cytokine, epidermal growth factor, fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor, an interferon, an interleukin, a keratinocyte growth factor, a leukemia inhibitory factor, oncostatin M, platelet derived erythroid colony stimulating factor (PD-ECSF), Platelet-derived growth factor (PDGF), pleiotropin, stem cell factor (SCF), c-kit ligand, vascular endothelial growth factor (VEGF), granulocyte-colony stimulating factor (G-CSF), an oncogene product, a tumor suppressor, a steroid hormone receptor, a plant hormone, a disease resistance gene, an herbicide resistance gene product, a bacterial gene product, a monooxygenase, a protease, a nuclease, and a lipase.

30. The method of claim 1, wherein the set of oligonucleotides comprises one or more oligonucleotide member of between about 20 and about 60 nucleotides in length.

31. The method of claim 1, wherein the selected recombinant nucleic acid is selected for an in vivo selection assay or a parallel solid phase assay.

32. The method of claim 1, wherein the selected recombinant nucleic acid is selected for an in vitro selection assay.

33. The method of claim 1, further comprising deconvolution of the recombinant nucleic acid.

34. The method of claim 1, further comprising sequencing or cloning the selected recombinant nucleic acid.

35. The method of claim 1, wherein the set of oligonucleotides is synthesized in vitro by assembly PCR.

36. The method of claim 1, wherein the set of oligonucleotides is synthesized in vitro by error-prone assembly PCR.

37. The method of claim 1, wherein one or more of the parental character strings comprises a diplomat sequence.