Elimination of redundant and stop codons during the chemical synthesis of degenerate oligonucleotides. Combinatorial testing on the chromophore region of the red fluorescent protein mKate.

Although some strategies have been reported for the elimination of stop and redundant codons during the chemical synthesis of degenerate oligonucleotides, incorporating an expensive cocktail of 20 trimer-phosphoramidites is currently a commonly employed and straightforward approach. As an alternative option, we describe here a cheaper strategy based on standard monomer-phosphoramidites and a simplified resin-splitting procedure. The accurate division of the resin, containing the growing oligonucleotide, into four columns represents the key step in this approach. The synthesis of the degenerate codon NDT in column 1, loaded with 60% of the resin, produces 12 codons, while a degenerate codon VMA in column 2, loaded with 30% of the resin, produces 6 codons. Codons ATG and TGG, independently synthesized in columns 3 and 4, respectively, and loaded with 5% each, completes the 20 different codons. The experimental frequency of each mutant codon in the library was assessed by randomizing 12 contiguous codons that encode for amino acids located in the chromophore region of the enhanced red fluorescent protein mKate-S158A. Furthermore, randomization of three contiguous codons that encode for the amino acids Phe62, Met63, and Tyr64, which are equivalent to Phe64, Ser65, and Tyr66 in GFP, gave rise to some red and golden yellow fluorescent mutants displaying interesting phenotypes and spectroscopic properties. The absorption and emission spectra of two of these mutants also suggested that the complete maturation of the red and golden yellow chromophores in mKate proceeds via the formation of a green-type chromophore and a cyan-type chromophore, respectively.