High-throughput synthesis of DNA fragments by molecular self-assembly of overlapping oligonucleotides

Nature Biotechnology
(2026) Cite this article

The limitations of DNA synthesis technologies are a fundamental bottleneck in synthetic biology. Here we present a high-throughput gene synthesis method driven by hybridization, called Molecular Self-Assembly Induced Cloning. Molecular Self-Assembly Induced Cloning overcomes molecular crosstalk and oligo misalignment across genes by coupling orthogonal self-assembly of overlapping DNA segments in vitro with the DNA repair machineries of host cells in vivo. Cellular uptake of the assembled target fragments serves as templates for recovery and cloning. We adopt microchip-based oligonucleotide synthesis, enabling the production of over 1,000 distinct gene fragments in a simple one-pot reaction. Near-zero misalignment is an indispensable feature of the parallel synthesis, with oligo synthesis errors remaining at a constant but controllable level. We also construct massive variant libraries of the industrial enzyme PETase and discover higher-potency variants than the gold standard enzyme.

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The NGS datasets are available from NCBI with accession code PRJNA1470352 (ref. 37). Other data supporting the findings of this study are available within the paper and Supplementary Information. Source data are provided with this paper.

All codes used in this study are available via GitHub at https://github.com/zgwuthu/MOSAIC.git (ref. 38).

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