Periodic Reporting for period 3 - PRO-TOOLKITS (Programmable nucleic acid toolkits for cell-free diagnostics and genetically encoded biosensing)
Période du rapport: 2022-10-01 au 2024-03-31
WHAT: The goal of the PRO-TOOLKITS project is to address this need by developing innovative cell-free point of care diagnostic kits and genetically encodable biosensing tools.
WHY: The biological complexity of tumours and the large diversity of diagnostic biomarkers call for the development of innovative analytical tools that can detect multiple targets in a sensitive, specific and low-cost way and allow real-time monitoring of disease pathways and therapeutic effects. To provide such transformative tools creative thinking, innovative approach and the exploration of new research avenues that span different disciplines is necessary.
OVERALL OBJECTIVES:The key, ground-breaking IDEA underlying this project is to take advantage of my expertise and harness the advantageous features of RNA synthetic modules that can translate the expression of proteins in controlled in-vitro cell-free systems and can be also genetically encoded in living organisms and function inside the cells. I will develop rationally designed programmable nucleic acid modules that respond to a wide range of molecular markers and environmental stimuli through innovative nature-inspired mechanisms and that can be orthogonally wired to provide cell-free diagnostic kits and genetically encoded live-cell biosensing tools. The project will provide transformative approaches, methods and tools that will represent a genuine break-through in the fields of in-vitro diagnostics, biosensing and synthetic biology.
WHY: The biological complexity of tumours and the large diversity of diagnostic biomarkers call for the development of innovative analytical tools that can detect multiple targets in a sensitive, specific and low-cost way and allow real-time monitoring of disease pathways and therapeutic effects. To provide such transformative tools creative thinking, innovative approach and the exploration of new research avenues that span different disciplines is necessary.
OVERALL OBJECTIVES:The key, ground-breaking IDEA underlying this project is to take advantage of my expertise and harness the advantageous features of RNA synthetic modules that can translate the expression of proteins in controlled in-vitro cell-free systems and can be also genetically encoded in living organisms and function inside the cells. I will develop rationally designed programmable nucleic acid modules that respond to a wide range of molecular markers and environmental stimuli through innovative nature-inspired mechanisms and that can be orthogonally wired to provide cell-free diagnostic kits and genetically encoded live-cell biosensing tools. The project will provide transformative approaches, methods and tools that will represent a genuine break-through in the fields of in-vitro diagnostics, biosensing and synthetic biology.
The project is split into three major Objectives. During the course of the first reporting period I have tackled the first two major Objectives.
Objective 1 (O1): TRICKS: Recreate nature-inspired tricks using synthetic RNA.
Objective 2 (O2): MODULES: Create a library of programmable responsive nucleic acid modules.
For Objective 1 we have worked on the four aims planned in the original proposal. More specifically we have designed and re-engineered different RNA and DNA elements with intrinsically-disordered regions to control and regulate their function in response to pH and temperature. These re-engineered devices allow a precise control of strand displacement reactions using pH and temperature and might be used in conjunction with cell-free genetic circuits or self-assembly systems to control the transcription/translation of genetic circuits or the assembly/disassembly of nucleic acids structures.
For aim 1.2 we have worked on the rational design of RNA and DNA-based receptors controlled by templated reactions.
In a first effort we have reported a rational strategy to orthogonally control assembly and disassembly of DNA-based nanostructures using specific IgG antibodies as molecular inputs. We have first demonstrated that the binding of a specific antibody to a pair of antigen-conjugated split DNA input-strands induces their co-localization and reconstitution into a functional unit that is able to initiate a toehold strand displacement reaction. The effect is rapid and specific and can be extended to different antibodies with the expedient of changing the recognition elements attached to the two split DNA input-strands. Such an antibody-regulated DNA-based circuit has then been employed to control the assembly and disassembly of DNA tubular structures using specific antibodies as inputs.
In a second effort we have also reported the rational design of DNA templated synthesis controlled by specific IgG antibodies. The approach is based on the co-localization of reactants induced by the bivalent binding of a specific IgG antibody to two antigen-conjugated DNA templating strands that triggers a chemical reaction that would be otherwise too slow under diluted conditions. This strategy is versatile, orthogonal and adaptable to different IgG antibodies and can be employed to achieve the targeted synthesis of clinically-relevant molecules in the presence of specific IgG biomarker antibodies.
In aim 1.3 we have studied and recreated the ultrasensitive mechanisms to control the dynamic response of RNA and DNA-based receptors. We are working on amplification strategies based on both enzymatic reactions and non-enzymatic reactions that will lead to ultrasensitive response.
Finally, for aim 1.4 we have re-programmed out-of-equilibrium mechanisms for the temporal control of RNA and DNA-based receptors.
In this case we have provided the first example of kinetically controlled DNA nanostructures in which energy dissipation is achieved through a non-enzymatic chemical reaction.
In another effort we also took advantage of the programmability of DNA/DNA interactions to report the rational design of orthogonal DNA-based addressable tiles that self-assemble into polymer-like structures that can be reconfigured by external inputs.
For Objective 2: MODULES we planned to create a library of programmable responsive nucleic acid modules. The idea was to develop a library of biomolecular responsive modules that respond to different inputs and stimuli and whose input/output behaviour can be easily programmed. The modules should also fused with the GFP-like RNA aptamer used in O1 (Spinach). This will allow to create a set of GFP-like RNA responsive modules.
This is ongoing work in the lab. We have first designed an electrochemical DNA circuit that responds quantitatively to multiple specific antibodies. The approach employs synthetic antigen-conjugated nucleic acid strands that are rationally designed to induce a strand displacement reaction and release a redox-reporter modified strand upon the recognition of a specific target antibody. The approach is sensitive (low nanomolar detection limit), specific (no signal is observed in the presence of non-targeted antibodies) and selective (the platform can be employed in complex media, including 90% serum).
We have also designed genetic cell-free circuits that respond to different antibodies and, in response, transcribe for a specific illuminating aptamer (mango or spinach). These results are still preliminary but we have obtained very promising data.
Objective 1 (O1): TRICKS: Recreate nature-inspired tricks using synthetic RNA.
Objective 2 (O2): MODULES: Create a library of programmable responsive nucleic acid modules.
For Objective 1 we have worked on the four aims planned in the original proposal. More specifically we have designed and re-engineered different RNA and DNA elements with intrinsically-disordered regions to control and regulate their function in response to pH and temperature. These re-engineered devices allow a precise control of strand displacement reactions using pH and temperature and might be used in conjunction with cell-free genetic circuits or self-assembly systems to control the transcription/translation of genetic circuits or the assembly/disassembly of nucleic acids structures.
For aim 1.2 we have worked on the rational design of RNA and DNA-based receptors controlled by templated reactions.
In a first effort we have reported a rational strategy to orthogonally control assembly and disassembly of DNA-based nanostructures using specific IgG antibodies as molecular inputs. We have first demonstrated that the binding of a specific antibody to a pair of antigen-conjugated split DNA input-strands induces their co-localization and reconstitution into a functional unit that is able to initiate a toehold strand displacement reaction. The effect is rapid and specific and can be extended to different antibodies with the expedient of changing the recognition elements attached to the two split DNA input-strands. Such an antibody-regulated DNA-based circuit has then been employed to control the assembly and disassembly of DNA tubular structures using specific antibodies as inputs.
In a second effort we have also reported the rational design of DNA templated synthesis controlled by specific IgG antibodies. The approach is based on the co-localization of reactants induced by the bivalent binding of a specific IgG antibody to two antigen-conjugated DNA templating strands that triggers a chemical reaction that would be otherwise too slow under diluted conditions. This strategy is versatile, orthogonal and adaptable to different IgG antibodies and can be employed to achieve the targeted synthesis of clinically-relevant molecules in the presence of specific IgG biomarker antibodies.
In aim 1.3 we have studied and recreated the ultrasensitive mechanisms to control the dynamic response of RNA and DNA-based receptors. We are working on amplification strategies based on both enzymatic reactions and non-enzymatic reactions that will lead to ultrasensitive response.
Finally, for aim 1.4 we have re-programmed out-of-equilibrium mechanisms for the temporal control of RNA and DNA-based receptors.
In this case we have provided the first example of kinetically controlled DNA nanostructures in which energy dissipation is achieved through a non-enzymatic chemical reaction.
In another effort we also took advantage of the programmability of DNA/DNA interactions to report the rational design of orthogonal DNA-based addressable tiles that self-assemble into polymer-like structures that can be reconfigured by external inputs.
For Objective 2: MODULES we planned to create a library of programmable responsive nucleic acid modules. The idea was to develop a library of biomolecular responsive modules that respond to different inputs and stimuli and whose input/output behaviour can be easily programmed. The modules should also fused with the GFP-like RNA aptamer used in O1 (Spinach). This will allow to create a set of GFP-like RNA responsive modules.
This is ongoing work in the lab. We have first designed an electrochemical DNA circuit that responds quantitatively to multiple specific antibodies. The approach employs synthetic antigen-conjugated nucleic acid strands that are rationally designed to induce a strand displacement reaction and release a redox-reporter modified strand upon the recognition of a specific target antibody. The approach is sensitive (low nanomolar detection limit), specific (no signal is observed in the presence of non-targeted antibodies) and selective (the platform can be employed in complex media, including 90% serum).
We have also designed genetic cell-free circuits that respond to different antibodies and, in response, transcribe for a specific illuminating aptamer (mango or spinach). These results are still preliminary but we have obtained very promising data.
The proposed research is extremely timely: the project will provide new biosensing tools to be applied for the detection of a wide range of targets going beyond the state of the art.
The project will thus have a great impact in the fields of point-of-care diagnostics and imaging providing the following novel concepts and approaches:
Cell-free systems: synthetic cell-free networks might be the future for diagnostic applications. These systems are versatile, low-cost, can be very sensitive and in principle adaptable to the detection of any target.
Genetically-encoded RNA imaging tools: the demonstration of GFP-like RNA systems have represented one of the most exciting innovations in the last years. I will contribute to this emerging field by providing new tools that can respond to a wide range of inputs opening to new opportunities of application.
The project will thus have a great impact in the fields of point-of-care diagnostics and imaging providing the following novel concepts and approaches:
Cell-free systems: synthetic cell-free networks might be the future for diagnostic applications. These systems are versatile, low-cost, can be very sensitive and in principle adaptable to the detection of any target.
Genetically-encoded RNA imaging tools: the demonstration of GFP-like RNA systems have represented one of the most exciting innovations in the last years. I will contribute to this emerging field by providing new tools that can respond to a wide range of inputs opening to new opportunities of application.