When synthetic biologists sketch gene circuits, they usually think in terms of promoters, repressors, and transcription factors—biochemical parts that toggle genes on or off. But DNA is not a flat schematic. It’s a physical polymer that twists, coils, and buckles as genes are transcribed. A pair of new papers from MIT and collaborators shows that this physicality could suggest approaches to controlling the output of gene circuits.

In a recent Science study titled “Gene syntax defines supercoiling-mediated transcriptional feedback,” researchers demonstrate that the order and orientation of neighboring genes—what they call gene syntax—can reshape local DNA supercoiling and, in turn, amplify or suppress the expression of adjacent genes.

“Syntax will be really useful for dynamic circuits. Now we have the ability to select not only the biochemistry of circuits, but also the physical design to support dynamics,” said Katie Galloway, PhD, an assistant professor of chemical engineering at MIT.

The team engineered human cell lines and hiPSCs with synthetic two‑gene reporter circuits arranged in tandem, divergent, or convergent configurations. Their earlier modeling predicted that divergent syntax should boost the expression of both genes, while tandem syntax should suppress the downstream gene. “The thing that we were trying to solve in this paper was: When you put two genes on the same piece of DNA, how does their physical interaction become coupled?” said Galloway. The experimental results matched those predictions: divergent circuits amplified both genes, while tandem circuits showed strong upstream‑to‑downstream repression, with effects reaching up to 25‑fold.

To understand why, the researchers used Region Capture Micro‑C, a high‑resolution genome‑folding mapping technique, to visualize how transcription reshapes DNA. When a gene was activated, the DNA downstream tightened into plectonemes—twisted structures that hinder RNA polymerase binding—while upstream DNA loosened. “Supercoiling impacts transcription of adjacent genes by altering RNA polymerase binding, forming a feedback loop,” the authors of the first paper wrote.

The second paper, published in Nature Biomedical Engineering and titled “STRAIGHT-IN Dual: a platform for dual single-copy integrations of DNA payloads and gene circuits into human induced pluripotent stem cells,” introduced STRAIGHT‑IN Dual, a platform that enables simultaneous, allele‑specific, single‑copy integration of two DNA constructs into hiPSCs. This system allowed the team to “investigate how promoter choice and gene syntax influence transgene silencing and how these design features affect reporter expression and forward programming of hiPSCs into neurons, motor neurons, and endothelial cells,” according to the authors of the second paper.

Using STRAIGHT‑IN Dual, the researchers also demonstrated a practical application: a divergent circuit expressing two components of a yellow fever antibody produced higher output than other configurations.

“This is really exciting because we can coordinate gene expression in ways that just weren’t possible before,” Galloway said. “Now that we understand the syntax, I think this will pave the way for us to program dynamic behaviors.

“If you want coordinated expression, a divergent circuit is great. If you want something that’s either/or, you can imagine using a convergent or tandem circuit, so when one turns on, the other turns off, and you can alternate pulses,” Galloway added.

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