Circular ssDNA Opens A New Programming Layer For Cell-Free Gene Expression

By Friedrich C. Simmel, Ph.D., professor, Technical University of Munich

Synthetic biology has long depended on a familiar toolkit for cell-free protein expression (CFE): double-stranded DNA templates for transcription and RNA-based regulators for post-transcriptional control. Both approaches have enabled remarkable progress in biosensing, diagnostics, and biological computation. Yet each comes with limitations. DNA-based systems can be difficult to scale into highly programmable regulatory architectures, while RNA-based systems often struggle with instability and degradation.

My team recently explored a different possibility: What if circular single-stranded DNA (CssDNA) could function not merely as a structural material for DNA nanotechnology but as a programmable gene expression vector in cell-free systems?

The answer, surprisingly, was yes.

CssDNA has already become a foundational material in DNA origami and nanotechnology because of its addressability, programmability, and ability to form precisely engineered structures. However, its role in synthetic biology remained largely unexplored. Our team set out to determine whether CssDNA could actively participate in transcription and translation processes within a yeast-based CFE platform.

What we discovered was not only that CssDNA supports protein expression but that it introduces entirely new routes for gene regulation.

We engineered two CssDNA vectors containing a T7 promoter and an EGFP reporter gene: a sense strand configuration, CssDNA(+), and an antisense configuration, CssDNA(−). Both successfully produced protein inside the cell-free system, but they behaved very differently mechanistically. CssDNA(+) relied primarily on complete DNA replication before transcription could proceed. CssDNA(−), however, revealed something more interesting: it could access dual expression pathways.

At higher concentrations, CssDNA(−) was capable of initiating transcription directly from partially hybridized intermediates, without requiring full double-stranded DNA synthesis. In other words, incomplete replication products could already become transcriptionally active. This represented a fundamentally different mode of gene regulation from conventional plasmid-based systems.

The promoter region proved especially important. By introducing short complementary DNA strands targeting the T7 promoter, we could dramatically enhance or suppress expression. Even partially double-stranded promoter regions containing as little as nine accessible base pairs enabled transcriptional activation. This showed that CssDNA could be dynamically regulated through programmable hybridization events rather than relying solely on traditional transcription factors.

That flexibility creates exciting opportunities for synthetic biology.

Because CssDNA is inherently programmable, it becomes possible to integrate concepts from DNA nanotechnology directly into gene expression systems. Strand displacement reactions, secondary structure engineering, and sequence-addressable hybridization can all be used to modulate transcriptional behavior in predictable ways.

To demonstrate this capability, we constructed several logic gates — including OR, NOR, INHIBIT, NAND, and AND architectures — using CssDNA(−) as the regulatory core. In these systems, DNA strands functioned as inputs while protein fluorescence served as the output. By selectively enabling or blocking promoter accessibility, we could precisely control expression states and improve signal-to-noise ratios through manipulation of replication pathways.

Importantly, this work suggests that CssDNA is not simply another alternative vector format. It represents a bridge between two previously distinct disciplines: synthetic biology and DNA nanotechnology.

For years, DNA nanotechnology has excelled at building programmable structures and dynamic molecular devices. Synthetic biology, meanwhile, has focused on engineering gene circuits and biological computation. CssDNA offers a way to merge these strengths into a unified platform where molecular architecture and gene regulation become tightly integrated.

The implications extend beyond logic gates. CssDNA-based systems could support next-generation biosensors, programmable diagnostics, responsive therapeutic platforms, and increasingly sophisticated artificial cellular systems. Because these vectors can be engineered structurally as well as genetically, they introduce an additional layer of control unavailable in conventional plasmid systems.

One particularly compelling aspect is scalability. Traditional transcription-factor-based regulation can become difficult to expand into large multilayered circuits because of limited orthogonal regulators. CssDNA, by contrast, can leverage the enormous design space of DNA hybridization and strand displacement chemistry, offering potentially far richer programmability.

Our findings also deepen our understanding of how noncanonical nucleic acid architectures behave inside biological expression environments. The observation that partially replicated CssDNA intermediates can directly support transcription raises intriguing questions about the interface between replication machinery, promoter accessibility, and polymerase recognition.

Ultimately, we view this work as an expansion of the synthetic biology toolbox: one that brings structural programmability into direct conversation with gene expression.

Cell-free systems are increasingly important for rapid prototyping, diagnostics, distributed manufacturing, and biological computation. As these applications grow more sophisticated, the need for highly programmable modular regulatory systems will only increase. CssDNA may offer a versatile new foundation for meeting that challenge.

By combining the precision of DNA nanotechnology with the functional output of synthetic biology, we believe programmable CssDNA vectors could help define the next generation of cell-free engineering platforms.

About The Author:

Friedrich C. Simmel, Ph.D., is a biophysicist and professor at the Technical University of Munich (TUM). He is a researcher in the field of DNA nanotechnology and is best known for his work on DNA nanomachines and dynamic DNA-based systems.

Simmel received a doctorate in experimental physics from LMU Munich in 1999. From 2000 to 2002, he was a post-doctoral researcher at Bell Labs. Simmel joined the TUM faculty as a full professor in 2007.