Mutagenesis using trinucleotide beta-cyanoethyl phosphoramidites.

ABSTRACT

There is no easy way to selectively introduce mixtures of codon triplets into mutagenesis libraries. Solid-phase-supported DNA synthesis using successive coupling of mixtures of mononucleotides can be made to supply 32 codons, which gives redundancies in coding for 20 natural amino acids, as well as an often unwanted stop codon. Resin-splitting methods have been described, but the representation of all permutations is limited by mechanical factors for a large library, and the method is experimentally cumbersome. To demonstrate a third, improved method, the 3'-cyanoethyl phosphoramidite codon triplets dATA, dCTT, dATC, dATG and dAGC were made by solution-phase methods, with protecting groups fully compatible with modern automated phosphoramidite DNA synthesis chemistry. The reagents were then used to synthesize a 54-mer DNA fragment, wherein 15 internal base pairs were randomized by coupling a mixture of the five codons five times. The fragment was amplified as a cDNA pool, which was subcloned into a phagemid vector, and 16 randomly selected recombinants from this mini-library were sequenced. These clones showed random incorporation of the proper transcribed codon sequences at the correct location. Other functional tests involving the trinucleotide phosphoramidites showed modest (ca. 70%) coupling efficiencies and structural integrity of the DNA produced.