Methods for identifying sets of oligonucleotides for use in an in vitro recombination procedures

• US 7,904,249 B2
• Filed: 10/31/2007
• Issued: 03/08/2011
• Est. Priority Date: 01/19/1999
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis Mayflower Holding LLC
• Inventors: del Cardayre, Stephen, Tobin, Matthew, Patten, Phillip A., Giver, Lorraine J., Minshull, Jeremy, Selifonov, Sergey A., Gustafsson, Claes, Stemmer, Willem P. C.
• Primary Examiner(s): Zhou; Shubo (Joe)
• Application Number:
  US11/982,405
• Publication Number:
  US 20080318795A1
• Time in Patent Office: 1,224 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 are used to identify sets of oligonucleotides for in vitro recombination. In certain embodiments, the methods involve inputting amino acid sequence character strings into a digital system and back-translating the amino acid character strings into a plurality of nucleic acid character strings. The nucleic acid character strings are then processed to define and select at least one recombinant nucleic acid for use in identifying one or more oligonucleotides for in vitro recombination.

Claims

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

(a) inputting a plurality of amino acid sequence character strings into a digital system;

(b) back-translating the plurality of amino acid character strings in the digital system into a plurality of nucleic acid character strings;

(c) selecting certain back-translated nucleic acid character strings from the plurality of nucleic acid character strings based on species codon bias in a selected expression host or optimized sequence similarity between the plurality of nucleic acid character strings;

(d) selecting one or more cross-over sites on the selected nucleic acid character strings, thereby defining one or more recombinant nucleic acids that result from cross-overs between the selected nucleic acid character strings at the one or more cross-over sites;

(e) selecting at least one of the recombinant nucleic acids; and

(f) identifying one or more oligonucleotides for in vitro recombination by choosing at least one portion of at least one portion of at least one of the recombinant nucleic acids selected in (e) which at least one portion corresponds to the one or more oligonucleotides identified for in vitro recombination,

wherein at least operations (a) through (d) and (f) are performed via a suitably programmed computer system.

2. The method of claim 1, wherein selecting one or more cross over points is based on structural information comprising information depicting the three-dimensional structure of at least a portion of amino acids corresponding to the plurality of amino acid sequence character strings.

3. The method of claim 1, wherein (d) comprises selecting cross-over points that correspond to overlapping nucleic acid character sub-strings in the selected nucleic acid character strings.

4. The method of claim 1, wherein (d) comprises selecting cross-over points at sites that will preserve selected subunits, domains, or motifs in the plurality of amino acid sequence character strings.

5. The method of claim 1, wherein (d) comprises selecting cross-over points at sites chosen to maintain or disrupt one or more structural relationships between two or more amino acids represented by the plurality of amino acid sequence character strings.

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

7. The method of claim 6, wherein the genetic operation is selected from the group consisting of multiplication, mutation, fragmentation, and ligation.

8. The method of claim 1, wherein two or more amino acids corresponding to the plurality of amino acid sequence character strings comprise naturally occurring amino acids.

9. The method of claim 1, wherein (e) comprises assessing three-dimensional structural stability of at least some of amino acids corresponding to the plurality of amino acid sequence character strings.

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

bridging”

oligonucleotides.

11. The method of claim 1, further comprising aligning two or more nucleic acid character strings from the plurality of nucleic acid character strings prior to selecting one or more cross-over sites on nucleic acid character strings from the plurality of nucleic acid character strings.

12. The method of claim 1, wherein the recombinant nucleic acids that result from the cross-overs comprise contiguous sequences of multiple nucleotides from the plurality of nucleic acid character strings.

13. A computer program product comprising a machine readable storage 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) inputting a plurality of amino acid sequence character strings into a digital system;

(b) code for back-translating the plurality of amino acid character strings in the digital system into a plurality of nucleic acid character strings,

(c) code for selecting certain back-translated nucleic acid character strings from the plurality of nucleic acid character strings based on species codon bias in a selected expression host or optimized sequence similarity between the plurality of nucleic acid character strings;

(d) code for selecting one or more cross-over sites on the selected nucleic acid character strings, thereby defining one or more recombinant nucleic acids that result from cross-overs between the selected nucleic acid character strings at the one or more cross-over sites;

(e) code for selecting at least one of the recombinant nucleic acids; and

(f) code for identifying one or more oligonucleotides for in vitro recombination by choosing at least one portion of at least one portion of at least one of the recombinant nucleic acids selected in (d) 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 selecting one or more cross over points is based on structural information comprising information depicting the three-dimensional structure of at least a portion of amino acids corresponding to the plurality of amino acid sequence character strings.

15. The computer program product of claim 13, wherein (d) comprises code for selecting cross-over points that correspond to overlapping nucleic acid character sub-strings in the plurality of nucleic acid character strings.

16. The computer program product of claim 13, wherein (d) comprises code for selecting cross-over points at sites that will preserve selected subunits, domains, or motifs in the plurality of amino acid sequence character strings.

17. The computer program product of claim 13, wherein (d) 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 represented by the plurality of amino acid sequence character strings.

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

19. The computer program product of claim 13, 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 two or more amino acids corresponding to the plurality of amino acid sequence character strings comprise naturally occurring amino acids.

21. The computer program product of claim 13, wherein (e) comprises code for assessing three-dimensional structural stability of at least some of amino acids corresponding to the plurality of amino acid sequence character strings.

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

bridging”

oligonucleotides.

23. The computer program product of claim 13, further comprising code for aligning two or more nucleic acid character strings from the plurality of nucleic acid character strings prior to selecting one or more cross-over sites on nucleic acid character strings from the plurality of nucleic acid character strings.

24. The computer program product of claim 13, wherein the recombinant nucleic acids that result from the cross-overs comprise contiguous sequences of multiple nucleotides from the plurality of nucleic acid character strings.