TrimerDimer: an oligonucleotide-based saturation mutagenesis approach that removes redundant and stop codons

Abstract

9-fluorenylmethoxycarbonyl (Fmoc) and 4,4'-dimethoxytrityl (DMTr) are orthogonal hydroxyl protecting groups that have been used in conjunction to assemble oligonucleotide libraries whose variants contain wild-type and mutant codons randomly interspersed throughout a focused DNA region. Fmoc is labile to organic bases and stable to weak acids, whereas DMTr behaves oppositely. Based on these chemical characteristics, we have now devised TrimerDimer, a novel codon-based saturation mutagenesis approach that removes redundant and stop codons during the assembly of degenerate oligonucleotides. In this approach, five DMTr-protected trinucleotide phosphoramidites (dTGG, dATG, dTTT, dTAT and dTGC) and five Fmoc-protected dinucleotide phosphoramidites (dAA, dTT, dAT, dGC and dCG) react simultaneously with a starting oligonucleotide growing on a solid support. The Fmoc group is then removed and the incorporated dimers react with a mixture of three DMTr-protected monomer phosphoramidites (dC, dA and dG) to produce 15 trinucleotides: dCAA, dAAA, dGAA, dCTT, dATT, dGTT, dCAT, dAAT, dGAT, dCGC, dAGC, dGGC, dCCG, dACG and dGCG. After one mutagenic cycle, 20 codons are generated encoding the 20 natural amino acids. TrimerDimer was tested by randomizing the four contiguous codons that encode amino acids L64-G67 of an engineered, nonfluorescent GFP protein. Sequencing of 89 nonfluorescent mutant clones and isolation of two fluorescent mutants confirmed the principle.

Scheme 1.

Synthetic pathway for the preparation of DMTr-protected trimer phosphoramidites (DMTr-dXYZp). (A) Synthesis of 3′ Fmoc-protected deoxynucleosides. (B) Synthesis of DMTr-protected trinucleotides (DMTr-dXYZ) by the phophitetriester method in solution-phase. (C) Final phosphitylation. X, Y or Z: any of the bases properly protected in the exocyclic amino group. The abbreviations are described in ‘Materials and Methods’ section.

Figure 1.

TrimerDimer mutagenesis approach. (A) The mutagenic cycle begins with a 5′ de-protected oligonucleotide growing on a solid support and is composed of two couplings. In the first coupling, a mix of DMTr-trimers and Fmoc-dimers is reacted with the oligonucleotide in the presence of ETT as an activating reagent. A phosphoramidite-based capping step, an ordinary capping step and ordinary oxidation step complete this cycle. In the second coupling, the Fmoc group is removed by alkaline hydrolysis. The recently incorporated dimers are converted in trimers by the addition of three DMTr-protected monomers and ETT, the cycle is completed as the first coupling, the DMTr group is removed by acid hydrolysis and the mutagenic cycle can be repeated or ordinary synthesis can be resumed to assemble the 5′ wild-type flanking region. (B) Chemical structures of monomers, dimers and trimers employed in the TrimerDimer mutagenesis approach. In these compounds, cytosine and adenine are protected with the benzoyl group and guanine with the isobutyryl group. Thymine is not protected. (C) Codons and encoded amino acids generated by TrimerDimer mutagenesis. (D) DNA synthesizer implemented to automate TrimerDimer mutagenesis. All abbreviations are described in ‘Materials and Methods’ section.

Figure 2.

(A) Pure proteins sgGFPr, sgGFPr-C65A and sgGFPr-L64A/C65A photographed under sunlight. (B) Fluorescence excitation and emission spectra (solid and dashed lines, respectively) of the pure proteins sgGFPr (black), sgGFPr-C65A (blue) and sgGFPr-L64A/C65A (red).