Engineered eMutS based magnetic separation system for high-fidelity DNA synthesis

DNA synthesis fidelity remains a critical bottleneck in large-scale gene assembly. Although conventional post-synthetic purification methods (e.g., HPLC, PAGE) and enzymatic mismatch-cleavage strategies offer partial solutions, they often suffer from limited throughput, high cost, and incompatibility with automated workflows. To overcome these limitations, we developed an integrated, sensitive and high-throughput error-correction platform that combines a rationally engineered Escherichia coli MutS protein (eMutS) with a biotin-streptavidin magnetic-bead separation system (eMBS). Specifically, a single point mutation (E38N) was introduced into eMutS to enhance mismatch-recognition specificity while suppressing non-specific binding to perfectly matched DNA by optimizing key residue-base interactions of enhanced π-π stacking and direct hydrogen bond formation. Two cysteine mutations (L157C/G233C) were further incorporated to promote disulfide bond formation, which strengthens the affinity toward all three error types and enhances structural stability. The resulting eMutS-L157C/G233C-E38N-biotin fusion protein which was immobilized onto streptavidin-coated magnetic beads, enables rapid and efficient magnetic separation of error-containing heteroduplexes from error-free homoduplexes. In terms of superior performance with mixed DNA substrates, eMBS reduces total errors by 13.1∼63.3% (n = 3 independent experiments) in pools starting with 73.6∼98.6% correct sequences, and achieves an error-free DNA ratio of 99.1%. By eliminating labor-intensive electrophoresis or column purification steps, this sensitive, versatile and high-throughput workflow can support high-fidelity DNA synthesis.