Periodic Reporting for period 2 - iSenseDNA (Computation driven development of novel vivo-like-DNA-nanotransducers for biomolecules structure identification)
Periodo di rendicontazione: 2023-10-01 al 2025-03-31
The primary goal of iSenseDNA is to innovate the realm of bio-molecular sensing by crafting personalized DNA structures, essentially serving as "translators" to unveil concealed information related to bio-molecular processes.
The groundbreaking methodology spearheaded by iSenseDNA involves the utilization of "DNA nanotransducers" (DNA-NTs) and their dynamic interactions with proteins.
This pioneering approach has the potential to revolutionize medical diagnostics and treatment modalities.
To accomplish this overarching objective, the project is strategically broken down into the following intermediate goals:
A. Develop a comprehensive repertoire of enzymes featuring 'novel activities' to modify DNA structures.
B. Engineer and demonstrate tailored DNA-NTs covering a diverse spectrum of topologies pertinent to cellular processes. These DNA-NTs are also equipped with specific DNA sequence modifications to facilitate interactions with targeted proteins.
C. Attain an in-depth understanding of the interactions between DNA-NTs and proteins, closely monitoring the structural alterations in DNA when bound to proteins.
D. Validate and explore potential applications of DNA-NTs both before and after interactions with model proteins, such as GCN4 and alpha-synuclein.
These intermediate objectives collectively propel iSenseDNA's advancements in the field of bio-molecular sensing, laying the groundwork for groundbreaking developments in diagnostics and therapeutics.
This project stems from the recognition of pressing challenges and needs in bio-molecular research.
By creating custom DNA structures and utilizing innovative DNA-NTs, iSenseDNA aims to address the intricate details of cellular processes, with a particular focus on medical applications.
The project envisions contributing significantly to the field, offering solutions to identified problems in the context of evolving political and strategic landscapes.
The anticipated impact of iSenseDNA's results is substantial, as successful implementation could lead to transformative changes in medical diagnostics and therapeutic interventions.
The groundbreaking methodology spearheaded by iSenseDNA involves the utilization of "DNA nanotransducers" (DNA-NTs) and their dynamic interactions with proteins.
This pioneering approach has the potential to revolutionize medical diagnostics and treatment modalities.
To accomplish this overarching objective, the project is strategically broken down into the following intermediate goals:
A. Develop a comprehensive repertoire of enzymes featuring 'novel activities' to modify DNA structures.
B. Engineer and demonstrate tailored DNA-NTs covering a diverse spectrum of topologies pertinent to cellular processes. These DNA-NTs are also equipped with specific DNA sequence modifications to facilitate interactions with targeted proteins.
C. Attain an in-depth understanding of the interactions between DNA-NTs and proteins, closely monitoring the structural alterations in DNA when bound to proteins.
D. Validate and explore potential applications of DNA-NTs both before and after interactions with model proteins, such as GCN4 and alpha-synuclein.
These intermediate objectives collectively propel iSenseDNA's advancements in the field of bio-molecular sensing, laying the groundwork for groundbreaking developments in diagnostics and therapeutics.
This project stems from the recognition of pressing challenges and needs in bio-molecular research.
By creating custom DNA structures and utilizing innovative DNA-NTs, iSenseDNA aims to address the intricate details of cellular processes, with a particular focus on medical applications.
The project envisions contributing significantly to the field, offering solutions to identified problems in the context of evolving political and strategic landscapes.
The anticipated impact of iSenseDNA's results is substantial, as successful implementation could lead to transformative changes in medical diagnostics and therapeutic interventions.
iSenseDNA project has made substantial progress in advancing its scientific and technical objectives. Work has been carried out effectively across all work packages, leading to important achievements in enzyme engineering, DNA nanotechnology, structural modeling, and biophysical characterization.
Work centered on the design, production, and validation of novel topoisomerase variants has yielded promising results. Through a combination of computational modeling, simulation, and experimental validation, several engineered enzymes—and the variants—have demonstrated enhanced performance:
Redesigned enzymes with improved sequence specificity and thermal stability were successfully expressed and shown to induce modification of DNA structures activity.
Mechanistic insights and chimeric enzyme development are enhancing the versatility and functionality of the enzyme portfolio.
These efforts lay a robust foundation for a functional toolkit of engineered enzymes critical to DNA nanostructure modification.
Advanced computational approaches have been employed to explore the structural dynamics of DNA nanostructures (DNA-NTs) and their interactions with target proteins:
Quantum mechanical calculations, molecular dynamics simulations, and docking analyses have elucidated the conformational behavior of supercoiled DNA and its complexes.
Simulations of infrared (IR) spectra are supporting the identification of spectroscopic signatures for both DNA and protein components.
This theoretical backbone has enabled predictive modeling that supports the rational design of DNA-based systems and feeds directly into experimental validation workflows.
Considerable progress has been made in characterizing DNA-NTs and their interaction with proteins using biophysical and spectroscopic techniques:
DNA nanostructures were rationally designed with specific topologies and sequences, and their physical properties were assessed using techniques such as 2D gel electrophoresis, circular dichroism, and mass spectrometry.
Label-free biolayer interferometry (BLI) methods were developed and applied to investigate binding kinetics and interaction dynamics.
Model systems, such as GCN4-DNA interactions, were studied to explore topology-dependent recognition mechanisms.
Set-up and calibration of 2D IR spectroscopic methods were completed, with ongoing Raman and FTIR analyses enriching the dataset.
These activities have provided key insights into real-time biomolecular interaction mechanisms and validated core optical platforms for molecular sensing.
Initial validation of the platform in biologically relevant systems has been successfully initiated:
High-yield production of GCN4 protein enabled structural characterization through SAXS/WAXS, linking computational predictions to experimental outcomes.
A disease-relevant biological model—brain organoids carrying alpha-synuclein gene triplication—was established to test the diagnostic and sensing capabilities of the platform.
Critical quality control and advanced molecular analyses of alpha-synuclein species are ongoing, supporting the application of the platform for neurodegenerative disease research.
These results demonstrate the platform’s translational potential and confirm its applicability in complex biological environments.
Work centered on the design, production, and validation of novel topoisomerase variants has yielded promising results. Through a combination of computational modeling, simulation, and experimental validation, several engineered enzymes—and the variants—have demonstrated enhanced performance:
Redesigned enzymes with improved sequence specificity and thermal stability were successfully expressed and shown to induce modification of DNA structures activity.
Mechanistic insights and chimeric enzyme development are enhancing the versatility and functionality of the enzyme portfolio.
These efforts lay a robust foundation for a functional toolkit of engineered enzymes critical to DNA nanostructure modification.
Advanced computational approaches have been employed to explore the structural dynamics of DNA nanostructures (DNA-NTs) and their interactions with target proteins:
Quantum mechanical calculations, molecular dynamics simulations, and docking analyses have elucidated the conformational behavior of supercoiled DNA and its complexes.
Simulations of infrared (IR) spectra are supporting the identification of spectroscopic signatures for both DNA and protein components.
This theoretical backbone has enabled predictive modeling that supports the rational design of DNA-based systems and feeds directly into experimental validation workflows.
Considerable progress has been made in characterizing DNA-NTs and their interaction with proteins using biophysical and spectroscopic techniques:
DNA nanostructures were rationally designed with specific topologies and sequences, and their physical properties were assessed using techniques such as 2D gel electrophoresis, circular dichroism, and mass spectrometry.
Label-free biolayer interferometry (BLI) methods were developed and applied to investigate binding kinetics and interaction dynamics.
Model systems, such as GCN4-DNA interactions, were studied to explore topology-dependent recognition mechanisms.
Set-up and calibration of 2D IR spectroscopic methods were completed, with ongoing Raman and FTIR analyses enriching the dataset.
These activities have provided key insights into real-time biomolecular interaction mechanisms and validated core optical platforms for molecular sensing.
Initial validation of the platform in biologically relevant systems has been successfully initiated:
High-yield production of GCN4 protein enabled structural characterization through SAXS/WAXS, linking computational predictions to experimental outcomes.
A disease-relevant biological model—brain organoids carrying alpha-synuclein gene triplication—was established to test the diagnostic and sensing capabilities of the platform.
Critical quality control and advanced molecular analyses of alpha-synuclein species are ongoing, supporting the application of the platform for neurodegenerative disease research.
These results demonstrate the platform’s translational potential and confirm its applicability in complex biological environments.
The platform envisioned in this project represents an innovative approach to constructing essential components and seamlessly integrating them. The primary aim is to create a unique sensing technology that implements advanced real-time diagnostics, enabling the identification and correlation of molecular structures and biological functions within a single system. This pioneering approach holds the potential to unlock specific applications in the domains of diagnostics and drug discovery.
iSenseDNA has the potential to significantly impact several scientific areas and markets, including:
(i) Bioinformatics and advanced computational methods, encompassing molecular dynamics at both classical and quantum levels, as well as machine learning.
(ii) Biotechnology, with a particular focus on protein engineering and molecular biology.
(iii) Advanced optical spectroscopy, including micro- and nanophotonics.
(iv) Nano-fabrication, specifically for micro/nano photonic chip development.
(v) Nanomedicine, exploring the application of nanoscale materials in medical contexts.
In comparison to the existing state-of-the-art in structural biology and diagnostics, iSenseDNA introduces the integration of costume-made DNA-NTs with photonic chips. This integration provides insights of DNA-NTs structural features, both independently and in the presence of interacting molecules.
The comprehensive integration proposed by iSenseDNA is expected to have two significant effects:
-It will contribute to and influence the European and global biotechnology, pharmaceutical, and sensor sectors, thereby elevating technological excellence and expanding the product offerings of EU entities, thereby strengthening their international position.
-It will enhance competitiveness, leading to advancements in the fields and providing substantial socio-economic benefits.
iSenseDNA has the potential to significantly impact several scientific areas and markets, including:
(i) Bioinformatics and advanced computational methods, encompassing molecular dynamics at both classical and quantum levels, as well as machine learning.
(ii) Biotechnology, with a particular focus on protein engineering and molecular biology.
(iii) Advanced optical spectroscopy, including micro- and nanophotonics.
(iv) Nano-fabrication, specifically for micro/nano photonic chip development.
(v) Nanomedicine, exploring the application of nanoscale materials in medical contexts.
In comparison to the existing state-of-the-art in structural biology and diagnostics, iSenseDNA introduces the integration of costume-made DNA-NTs with photonic chips. This integration provides insights of DNA-NTs structural features, both independently and in the presence of interacting molecules.
The comprehensive integration proposed by iSenseDNA is expected to have two significant effects:
-It will contribute to and influence the European and global biotechnology, pharmaceutical, and sensor sectors, thereby elevating technological excellence and expanding the product offerings of EU entities, thereby strengthening their international position.
-It will enhance competitiveness, leading to advancements in the fields and providing substantial socio-economic benefits.