Periodic Reporting for period 4 - PRO-TOOLKITS (Programmable nucleic acid toolkits for cell-free diagnostics and genetically encoded biosensing)
Période du rapport: 2024-04-01 au 2025-09-30
The PRO-TOOLKITS ERC Consolidator project addressed the urgent need for versatile, sensitive, and low-cost diagnostic technologies capable of detecting multiple disease markers and monitoring biological processes in real time, with a focus on cancer and immune-related conditions. By developing programmable nucleic acid modules and cell-free biosensing platforms, the project achieved its overall objective of creating innovative point-of-care diagnostic kits and genetically encodable tools that operate both in vitro and inside living cells.
Problem and context
Tumours and complex diseases are characterized by high biological heterogeneity and by a large variety of diagnostic biomarkers, which current clinical assays cannot monitor simultaneously, rapidly, and at low cost. This limitation hinders early diagnosis, stratification of patients, and real-time monitoring of therapeutic responses, especially in decentralized or resource-limited settings. There is therefore a critical need for analytical platforms that can integrate nucleic-acid programmability, responsiveness to diverse molecular cues, and compatibility with complex samples, while remaining modular and easy to adapt to new biomarkers.
Importance for society
The technologies developed in PRO-TOOLKITS contribute to advancing point-of-care diagnostics and biosensing by enabling sensitive, specific and multiplexed detection of antibodies, antigens and other molecular targets directly in complex matrices such as serum. Such platforms can support earlier and more accurate disease detection, facilitate personalized therapy monitoring, and ultimately reduce healthcare costs and improve patient outcomes. Beyond diagnostics, the ability of controlling nucleic-acid-based circuits and materials provide building blocks for future smart therapeutics, responsive biomaterials, and life‑like synthetic systems, with broad impact in biomedicine and biotechnology.
Overall objectives
We set out to develop rationally designed programmable nucleic-acid modules that respond to a wide range of molecular markers and environmental stimuli and can be orthogonally wired into cell-free diagnostic kits and genetically encoded live-cell biosensing tools.
Problem and context
Tumours and complex diseases are characterized by high biological heterogeneity and by a large variety of diagnostic biomarkers, which current clinical assays cannot monitor simultaneously, rapidly, and at low cost. This limitation hinders early diagnosis, stratification of patients, and real-time monitoring of therapeutic responses, especially in decentralized or resource-limited settings. There is therefore a critical need for analytical platforms that can integrate nucleic-acid programmability, responsiveness to diverse molecular cues, and compatibility with complex samples, while remaining modular and easy to adapt to new biomarkers.
Importance for society
The technologies developed in PRO-TOOLKITS contribute to advancing point-of-care diagnostics and biosensing by enabling sensitive, specific and multiplexed detection of antibodies, antigens and other molecular targets directly in complex matrices such as serum. Such platforms can support earlier and more accurate disease detection, facilitate personalized therapy monitoring, and ultimately reduce healthcare costs and improve patient outcomes. Beyond diagnostics, the ability of controlling nucleic-acid-based circuits and materials provide building blocks for future smart therapeutics, responsive biomaterials, and life‑like synthetic systems, with broad impact in biomedicine and biotechnology.
Overall objectives
We set out to develop rationally designed programmable nucleic-acid modules that respond to a wide range of molecular markers and environmental stimuli and can be orthogonally wired into cell-free diagnostic kits and genetically encoded live-cell biosensing tools.
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.
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.
During PRO-TOOLKITS we achieved and demonstrated a set of new technologies: dissipative DNA nanodevices and enzyme‑driven timers, antibody‑responsive circuits for nanostructure control and templated chemistry, electrochemical and cell‑free biosensors, and CRISPR‑based diagnostic assays that went far beyond the state of the art. Many of these results have been published in high‑impact journals, consolidating the PI’s leadership in DNA nanotechnology, synthetic biosensing and cell‑free diagnostics and contributing to the international visibility of the host institution.
Overall, the project not only delivered breakthrough scientific results but also generated impact through international networking and dissemination of the results and technologies.
Overall, the project not only delivered breakthrough scientific results but also generated impact through international networking and dissemination of the results and technologies.