Periodic Reporting for period 1 - Entropic DNA Sensors (Entropically programmable DNA-based bioSensors for personalized medicine)
Période du rapport: 2021-05-01 au 2023-04-30
Biosensors have achieved a significant feat in bioanalytical chemistry and translational science: high-frequency, real-time and quantitative measurements of clinically relevant molecules in vitro and in vivo. Despite their advantages, our ability to precisely control and regulate the binding activity of their bioreceptors (i.e. the recognition element) still represents a highly relevant bioengineering challenge and limitation. Indeed, complete control of the binding properties of bioreceptors would allow the design of new biosensors with improved and predictable analytical performance. These improvements in bioreceptor properties have a direct impact and benefit on any biosensing technology, not only the newly developed but also the already established techniques. In fact, this will allow the development of innovative, high performance and programmable biosensors capable of achieving personalized medicine. For example, the high precision of the measurements could allow the tailoring of dietary or drug therapy, improving the clinical treatment of cancer, diabetes and metabolic disorders. In the MSCA action (Entropic DNA Sensors), the experienced researcher (Dr. Andrea Idili) proposed a multidisciplinary, innovative and versatile approach that allowed to fine-tune the activity and the response of synthetic bioreceptors. Specifically, the work has focused on the exploration and characterization of various naturally inspired mechanisms, such as entropic allostery and sequestration, as a novel signal transduction mechanism to improve measurement precision. This has allowed the development of a general bioengineering approach to precisely tune biosensor performance through the rational introduction of intrinsically disordered moieties. Finally, the senior researcher demonstrated the application of these mechanisms in representative biosensors (electrochemical and optical) and their use for real-time, high-precision measurements of clinically relevant molecules to achieve the detection of clinically relevant molecules (i.e. doxorubicin, ATP, vancomycin, and NGAL) directly in complex biological fluids in vitro. To achieve these project goals, Dott. Idili's previous experience in in vivo biosensing and point-of-care (PoC) testing technologies was synergistically combined with the recognized expertise in functional DNA nanotechnology and synthetic biology of Prof. Francesco Ricci at the University of Rome Tor Vergata (UNITOV-Rome, Italy).
In the first phase of the action, the research activity was combined with the training plan for the experienced researcher. The aim was to provide the experienced researcher with a strong background in the design of intrinsically disordered domains and the use of optical techniques for their characterization. The acquired knowledge and experience was ultimately applied to the design of new intrinsically disordered aptamers (doxorubicin, ATP, methotrexate) where their binding activity can be fine-tuned. The acquired know-how was then used for the design and characterization of thermo-programmed synthetic DNA-based receptors (not yet reported) and for the development of an innovative simulative and experimental approach for the rational design of intrinsically disordered aptamer receptors.
In the next stage of the action, the rational control of the binding activity of the aptamer receptors has allowed the experienced researcher to characterize their thermodynamic and to develop a bioengineering approach for the rational introduction of disordered moieties. In addition, the close teamwork and collaboration with MSCA Fellow Dr. Alejandro Chamorro allowed the development and adaptation of a second naturally inspired mechanism (sequestration). As an entropic allostery, the sequestration mechanism also allows to program the analytical performance of the aptamer receptor. In addition, the training received on the thermodynamics (theoretical and experimental) of DNA-based receptors was applied to optimize the intrinsically disordered aptamer receptors on an electrochemical sensing platform. and validation of their novel signal transduction mechanism; and the use of the sequestration mechanism as an alternative naturally inspired approach to modulate the binding properties of EAB sensors. Finally, the training in theoretical modeling and biophysics of DNA-based receptors was applied to the development and validation of a theoretical model to unravel the effect of the poly(T) linker on the observed affinity of the intrinsically disordered receptors.
In a more advanced stage of the work, entropic allostery and sequestration mechanisms were taken into account to enhance the sensitivity and were applied in different biosensing platforms. Besides aptamer-based electrochemical sensors, they were applied to lateral flow assays (optical readout) and ELISA (optical readout) and tested in complex media, artificial urine, simulating a real scenario. In addition, entropic allostery has been applied to the development and dynamic range optimization of thermally programmed synthetic DNA-based receptors; and to the development of entropy-driven cell-free transcriptional sensors.
In the next stage of the action, the rational control of the binding activity of the aptamer receptors has allowed the experienced researcher to characterize their thermodynamic and to develop a bioengineering approach for the rational introduction of disordered moieties. In addition, the close teamwork and collaboration with MSCA Fellow Dr. Alejandro Chamorro allowed the development and adaptation of a second naturally inspired mechanism (sequestration). As an entropic allostery, the sequestration mechanism also allows to program the analytical performance of the aptamer receptor. In addition, the training received on the thermodynamics (theoretical and experimental) of DNA-based receptors was applied to optimize the intrinsically disordered aptamer receptors on an electrochemical sensing platform. and validation of their novel signal transduction mechanism; and the use of the sequestration mechanism as an alternative naturally inspired approach to modulate the binding properties of EAB sensors. Finally, the training in theoretical modeling and biophysics of DNA-based receptors was applied to the development and validation of a theoretical model to unravel the effect of the poly(T) linker on the observed affinity of the intrinsically disordered receptors.
In a more advanced stage of the work, entropic allostery and sequestration mechanisms were taken into account to enhance the sensitivity and were applied in different biosensing platforms. Besides aptamer-based electrochemical sensors, they were applied to lateral flow assays (optical readout) and ELISA (optical readout) and tested in complex media, artificial urine, simulating a real scenario. In addition, entropic allostery has been applied to the development and dynamic range optimization of thermally programmed synthetic DNA-based receptors; and to the development of entropy-driven cell-free transcriptional sensors.
Results achieved during the action include the design and characterization of thermo-programmed synthetic DNA-based receptors (not previously reported), the development of an innovative simulative and experimental approach for the rational design of intrinsically disordered aptamer receptors, the application of nature-inspired mechanisms (entropic allostery and sequestration) to tune the binding properties of receptors in biological assays. Additional efforts were focused on the development and validation of a theoretical model to elucidate the effect of the poly(T) linker on the observed affinity of the intrinsically disordered receptors and the development of innovative entropy-driven cell-free transcriptional sensors.