From the start of PRO-TOOLKITS to the end of the reporting period, the work was organized around three main objectives. Below we summarize the main results obtained for each objective.
Objective 1:
We designed and re‑engineered DNA and RNA elements that respond in a programmable way to environmental stimuli (pH, temperature) and biochemical/enzymatic inputs. Key results include:
- pH‑responsive nanoswitches enabling tunable pH‑dependent conformational changes.
- DNAzymes whose catalytic activity is precisely regulated by intrinsically disordered regions.
- Antibody‑templated DNA circuits controlling the assembly/disassembly of nanostructures via toehold‑mediated strand displacement, allowing orthogonal regulation of multiple nanostructures in the same solution.
- Antibody‑controlled DNA‑templated synthesis in which specific IgGs trigger reactions that would otherwise be too slow under dilute conditions.
The most transformative outcome was the systematic development of dissipative DNA nanotechnology, with fuel‑driven, kinetically controlled nanostructures that assemble, operate and disassemble in a time‑programmed fashion, establishing dissipative architectures as a route to life‑like behaviour in nucleic‑acid systems.
Objective 2:
We translated these design principles into functional biosensing modules for quantitative, selective detection of antibodies and other biomarkers, especially in complex clinical matrices. Using nucleic‑acid programmability, we built modular electrochemical and optical DNA circuits in which antigen‑conjugated strands undergo strand displacement and release a reporter upon target binding, achieving low‑nanomolar sensitivity, excellent specificity and robust performance in up to 90% serum. These results show that rationally designed nucleic‑acid circuits are generalizable sensing modules where the recognition element (antigen, aptamer or other ligand) can be exchanged while preserving a common signal‑transduction architecture.
Objective 3:
We then integrated responsive modules and sensing circuits into complete tools and demonstrators, with emphasis on cell‑free systems and point‑of‑care applications. Key results include:
- Electrochemical cell‑free biosensors that couple antigen‑decorated DNA gene circuits with a T7‑based transcription system and redox‑labelled reporters on disposable electrodes, enabling sensitive, selective and multiplexable detection directly in complex samples such as serum.
- Transcriptionally controlled nucleic‑acid receptors that convert gene‑expression signals into dynamic loading and release of molecular ligands.
- Transcriptional cascades in which sequential activation/deactivation of DNA building blocks controls the formation of distinct populations of self‑assembling polymers.
Together, these tools show how nucleic‑acid modules, transcriptional networks and CRISPR‑based components can be combined into powerful diagnostic devices that connect synthetic biology with real‑world biosensing.
Dissemination and communication activities were also integral to the project. Scientifically, the results were disseminated through numerous publications, invited talks and, in particular, the organization of international workshops on Functional DNA Nanotechnology, which served as focal events to showcase project achievements and foster community building around nucleic‑acid‑based technologies. These workshops strengthened international collaborations and positioned the PI’s group as a hub for functional DNA nanotechnology and synthetic biosensing.
Beyond the scientific community, media coverage contributed to raising public awareness of DNA nanotechnology, cell‑free diagnostics and CRISPR‑based biosensing, emphasizing their potential benefits for future healthcare. The ERC funding was instrumental in establishing and consolidating an independent research line, enabling the recruitment and mentoring of several postdoctoral researchers and PhD students who have since obtained competitive fellowships or permanent positions in academia and industry.
