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.
