Codon-Based Mutagenesis Design and Protocols

Codon-Based Mutagenesis Design / Protocol

Standard trimer codon phosphoramidite mixes are currently optimized around E. coli codon abundances, with the most abundant codon used to code for a given amino acid selected for inclusion. This optimization is reasonable, given that their expected use is for oligos slated for generation of E. coli-based libraries for protein engineering. Other organisms (e.g., yeast or Drosophila) have different codon abundances/usage patterns (Codon Usage in Different Organisms). Thus, when performing codon-directed mutagenesis in an organism other than E. coli, it is important to compare the codon usage pattern of that organism with that of E. coli to ascertain the suitability of the standard mixes.

After synthesis of a mutagenic oligonucleotide using Trimer Phosphoramidites, the oligo will have a randomized trimer codon region with fixed sequences on each side. For protein engineering applications, such mutagenic oligos typically are cloned into E. coli to construct a phage display library. The dut/ung method is one such construction method that is highly efficient in this regard (6).

Background: Dut is a gene in E. coli that encodes for dUTPase, which degrades dUTP. A dut(-) strain lacks this enzyme, so an elevated concentration of dUTP accumulates, resulting in incorporation of U in place of T at some base positions during DNA replication. The ung gene encodes uracil N-glycosylase, which normally excises U from DNA. A ung(-) strain lacks this, so U bases are not excised from DNA in the bacterium. Thus, in a dut(-)/ung(-) double mutant, any U bases that are incorporated into DNA during replication are not repaired. However, because U and T have identical base-pairing properties, the T-to-U conversion is not mutagenic.

(1) A dut(-)/ung(-) strain is infected with M13 phage, and a circularized dU-containing M13 ssDNA template is obtained.

(2) The mutagenic oligonucleotide is annealed to this template in vitro via the fixed regions of the oligo (the mismatched randomized trimer codon region itself does not bind to the template).

(3) T7 DNA polymerase and T4 ligase are added, and the mutagenic oligo now functions as a primer for in vitro DNA replication, as the complementary strand is generated and the ends ligated together. The result is covalently closed circular dsDNA (CCC-ds DNA), with a dU-containing strand and a non-dU-containing strand (which contains the randomized trimer codon region).

(4) The CCC-ds-DNA is transformed into ung(+) E. coli. Uracil N-glycosylase excises U from the dU-containing strand, leaving apyrimidinic sites in it, which are recognized and cut by endonucleases. Thus the dU-containing strand is preferentially destroyed, and only the mutant strand is replicated, leading to its enrichment.

References

(1) Neylon, C. Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution. Nucleic Acids Res. (2004), 32: 1448-1459.
(2) Kayushin, A., Korosteleva, M., Miroshnikov, A. Large-scale solid-phase preparation of 3’-unprotected trinucleotide phosphotriesters-precursors for synthesis of trinucleotide phosphoramidites. Nucleosides Nucleotides Nucleic Acids (2000), 19: 1967-1976.
(3) Randolph, J., Yagodkin, A., Azhayev, A., Mackie, H. Codon-based Mutagenesis. Nucleic Acids Symposium Series (2008), 52: 479.
(4) Krumpe, L.R.H., Schumacher, K.M., McMahon, J.B., Makowski, L. Mori, T. Trinucleotide cassettes increase diversity of T7 phage-displayed peptide library. BMC Biotechol. (2007), 7: 65-72.
(5) Levy, M., Ellington, A.D. Directed Evolution of Streptavidin Variants Using IVC. Chem. Biol. (2008), 15: 979-989.
(6) Sidhu, S.S., Lowman, H.B., Cunningham, B.C., Wells, J.A. Phage display for selection of novel binding peptides. Methods Enzymol, 2000, 328, 333-363.