Codon-Based Mutagenesis Applications

Codon-Based Mutagenesis Applications

As described in the Introduction, the main use of trimer phosphoramidites is for developing partially randomized gene libraries based on the concept of codon-based mutagenesis. Because trimer codons cover all 20 natural amino acids, their use avoids problems associated with random point, or saturating, mutagenesis. In a random point mutagenesis approach, because the use of single base mutations generated by error-prone PCR produces only about six amino acid substitutions on average, concurrent mutation of adjacent nucleotides must occur; this requirement often makes it very difficult to find a clone with the desired amino acid change. For example, in a 100 aa protein, the likelihood of converting Tyr (TAC) to Asn (AAC) is only 1/900, the likelihood of converting Tyr (TAC) to Met (ATG) is 1.37 x 10E-9 (3). In saturating mutagenesis, pools of degenerate oligos are used with N’s at the site(s) of the codon(s) to be mutated. However, because some amino acids are represented by higher numbers of codons than others (for example, six for Arg, one for Trp), there will be large differences in the relative amounts of oligos containing particular amino acid mutations, leading to substantial undesired codon bias. Also, about 5% of the oligos will contain stop codons that will cause improper chain termination (3). By contrast, use of trimer phosphoramidites allows the incorporation of an equimolar mix of all 20 natural amino acids, or any subset, into any position of a sequence. This advantage eliminates codon bias, presence of unwanted stop codons or frameshift mutations, and makes searching a clonal library for desired mutants much more efficient. Multiple amino acids within a protein can be modified at the same time while the overall tertiary structure is maintained. Trimer phosphoramidites have been used to develop phage display libraries with greater diversity (as much as 10x greater) than by traditional methods, with a high degree of amino acid uniformity (4). Trimer phosphoramidites were also used to maximize the diversity of a partially randomized library for use in developing streptavidin variants with altered specificities for desthiobiotin, a biotin analog, by directed evolution (5).

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.