Solid Phase Assembly of Fully Protected Trinucleotide Building Blocks for Codon-Based Gene Synthesis

Ruth Suchsland,Matthäus Janczyk,Bettina Appel,Sabine Müller

doi:10.3390/app9112199

Abstract

The use of pre-formed trinucleotides, representing codons of the 20 canonical amino acids, for oligonucleotide-directed mutagenesis offers the advantage of controlled randomization and generation of “smart libraries”. We here present a method for the preparation of fully protected trinucleotides on solid phase. The key issue of our strategy is the linkage of the starting nucleoside to the solid support via a traceless disulfide linker. Upon trinucleotide assembly, the disulfide bridge is cleaved under reducing conditions, and the fully protected trinucleotide is released with a terminal 3′-OH group.

Highlights

A major goal of molecular biotechnology is the evolution/development of enzymes with desired and improved properties for industrial or pharmaceutical application [1,2]
A possibility to overcome these problems is the use of trinucleotide building blocks (Figure 1), which represent codons of the canonical amino acids and permit the controlled synthesis of a fully or partially randomized gene library without stop codons or codon bias [6]
Due to the obvious advantages of solid phase synthesis over solution chemistry, we sought to develop a strategy that allows the assembly of fully protected trinucleotides on solid support

Summary

Introduction

A major goal of molecular biotechnology is the evolution/development of enzymes with desired and improved properties for industrial or pharmaceutical application [1,2]. On the contrary, directed evolution offers the possibility, via random mutagenesis, of generating large protein libraries, which are screened for functionality, such that detailed structural information is not required [2]. Combinatorial gene synthesis offers the option of restricting structural variations to residues of interest, i.e., those that are involved in a certain function such as catalysis. The generation of stop codons cannot be prevented, which leads to abortion fragments instead of the full-length enzymes [5]. A possibility to overcome these problems is the use of trinucleotide building blocks (Figure 1), which represent codons of the canonical amino acids and permit the controlled synthesis of a fully or partially randomized gene library without stop codons or codon bias [6]. We have previously developed a strategy for synthesis in solution of fully protected trinucleotide phosphoramidites [6]

Methods

Results

Conclusion