MUTAGENESIS USING TRINUCLEOTIDE BETA-CYANOETHYL PHOSPHORAMIDITES

There is no easy way to selectively introduce mixtures of codon triple ts into mutagenesis libraries. Solid-phase-supported DNA synthesis usi ng successive coupling of mixtures of mononucleotides can be made to s upply 32 codons, which gives redundancies in coding for 20 natural ami no acids, as well as an often unwanted stop codon. Resin-splitting met hods 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, th e 3'-cyanoethyl phosphoramidite codon triplets dATA, dCTT, dATC, dATG and dAGC were made by solution-phase methods,,vith protecting groups f ully compatible with modern automated phosphoramidite DNA synthesis ch emistry. The reagents were then used to synthesize a 54-mer DNA fragme nt, wherein 15 internal base pairs were randomized by coupling a mixtu re of the five codons five rimes. The fragment was amplified as a cDNA pool, which was subcloned into a phagemid vector and 16 randomly sele cted 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 trinucle otide phosphoramidites showed modest (ca. 70%) coupling efficiencies a nd structural integrity of the DNA produced.