Synthetic sequence entanglement augments stability and containment of genetic information in cells

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Science  09 Aug 2019:
Vol. 365, Issue 6453, pp. 595-598
DOI: 10.1126/science.aav5477

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Overlapping genes for synthetic biology

Overlapping genes yield multiple distinct proteins when translated in alternative reading frames of the same nucleotide sequence. Blazejewski et al. developed a computational algorithm to predict de novo sequence entanglement and experimentally generated functional synthetic overlapping genes. When a sequence of interest was co-encoded with an essential gene in a living bacterium, its evolutionary stability substantially increased. When a gene of interest was synthetically overlapped with a toxin gene, its horizontal gene transfer frequency between bacteria was strongly suppressed. This generalizable strategy for designing, building, and testing overlapping genes helps stabilize vertical gene evolution and restrict horizontal gene flow.

Science, this issue p. 595


In synthetic biology, methods for stabilizing genetically engineered functions and confining recombinant DNA to intended hosts are necessary to cope with natural mutation accumulation and pervasive lateral gene flow. We present a generalizable strategy to preserve and constrain genetic information through the computational design of overlapping genes. Overlapping a sequence with an essential gene altered its fitness landscape and produced a constrained evolutionary path, even for synonymous mutations. Embedding a toxin gene in a gene of interest restricted its horizontal propagation. We further demonstrated a multiplex and scalable approach to build and test >7500 overlapping sequence designs, yielding functional yet highly divergent variants from natural homologs. This work enables deeper exploration of natural and engineered overlapping genes and facilitates enhanced genetic stability and biocontainment in emerging applications.

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