Periodic Reporting for period 1 - Nanodevice (Nanostructure-based label-free biomolecular binding kinetics assay)
Période du rapport: 2018-04-01 au 2019-03-31
The aim of the ERC Proof of Concept Grant proposal "Nanodevice" was the design, construction, experimental validation, and tests for possible applications of a nanoscale device that can accurately measure the binding and unbinding kinetics of target molecules. While these goals were not fully achieved in the course of the project, several subprojects were successful, leading to a large gain in know-how and practical learnings which will serve to guide future DNA origami nanostructure design and may lead to patent applications through the university technology transfer office. It led to an improved understanding of potential customer needs and promising technology applications, as well as inspiring future research directions.
The advances made during the project are related to three features of the envisioned nanodevice.
(1) Targeting a wide range of molecules through rationally attached interaction molecules
(2) Measure the binding of the target molecules to the interaction molecules by an advanced read-out based on conformational changes
(3) The read-out is based on changes in the fluorescent signal
In order to develop the capability to place rationally engineered interactions on the DNA origami nanostructures, covalent linkage of reactive molecules e.g. proteins, small molecules to single-stranded DNA using different linking chemistries was investigated. Moreover, the purification of the resulting complexes was one of the main challenges during the project. Efficient protocols for both steps were developed, and the successful incorporation of the complexes into assembled DNA nanostructures was investigated. To accurately measure the influence of binding on conformational changes and reciprocal effects, the influence of the attachment to a large DNA-origami nanostructure on the association constants and binding/unbinding rates of molecular interactions was studied. Significant influences, most likely due to the complex local environment of the DNA-origami nanostructure, were discovered, and will play an important role in future design considerations. In collaboration with a different project focused on the reverse effect of measuring the influence of conformational changes on the binding of target molecules, binding of DNA origami nanostructures as placeholders for target molecules was found to affect the conformation of a DNA-origami nanostructure, supporting the validity of the fundamental principle of the nanodevice.
In order to measure the binding of molecules and DNA-origami nanostructures to other DNA-origami nanostructures using a fluorescent readout, fluorescence polarization (FP) and Förster resonance energy transfer (FRET) assays were tested. FP signal to noise ratios were too low for effective use, but plate-reader based FRET measurements were successfully applied to the system, which will be useful in further high-throughput developments.
Another important result was found during attempts to precisely engineer the binding behavior of the conformational space of the DNA origami nanostructure. It was found that simple entropic chains, in the shape of single-stranded DNA strands, do not provide sufficient control over the conformation of the molecule. A combination with other interaction elements such as intermediate rigid double-stranded DNA strands as well as stacking interactions is required. The research on the validation of the binding behavior using these more complex building blocks was started in the course of the project, and is being continued in ongoing other research projects. The use of these building blocks for binding studies may also be evaluated for future patent applications in collaboration with the university technology transfer office, so that the nanodevice may contribute to future IP generation.
Concurrently with the described research work, literary research and discussions with experts in the field and key opinion leaders lead to the identification of proteins as the most promising field for targets for a potential functional nanodevice as well as for other DNA-origami nanostructure-based applications. Based on this, significant know-how on possible therapeutic targets, indications, manufacturers, researchers, and drugs in all stages of clinical development was established. This accumulation of information and expertise will be of great value for subsequent research projects on an even
The advances made during the project are related to three features of the envisioned nanodevice.
(1) Targeting a wide range of molecules through rationally attached interaction molecules
(2) Measure the binding of the target molecules to the interaction molecules by an advanced read-out based on conformational changes
(3) The read-out is based on changes in the fluorescent signal
In order to develop the capability to place rationally engineered interactions on the DNA origami nanostructures, covalent linkage of reactive molecules e.g. proteins, small molecules to single-stranded DNA using different linking chemistries was investigated. Moreover, the purification of the resulting complexes was one of the main challenges during the project. Efficient protocols for both steps were developed, and the successful incorporation of the complexes into assembled DNA nanostructures was investigated. To accurately measure the influence of binding on conformational changes and reciprocal effects, the influence of the attachment to a large DNA-origami nanostructure on the association constants and binding/unbinding rates of molecular interactions was studied. Significant influences, most likely due to the complex local environment of the DNA-origami nanostructure, were discovered, and will play an important role in future design considerations. In collaboration with a different project focused on the reverse effect of measuring the influence of conformational changes on the binding of target molecules, binding of DNA origami nanostructures as placeholders for target molecules was found to affect the conformation of a DNA-origami nanostructure, supporting the validity of the fundamental principle of the nanodevice.
In order to measure the binding of molecules and DNA-origami nanostructures to other DNA-origami nanostructures using a fluorescent readout, fluorescence polarization (FP) and Förster resonance energy transfer (FRET) assays were tested. FP signal to noise ratios were too low for effective use, but plate-reader based FRET measurements were successfully applied to the system, which will be useful in further high-throughput developments.
Another important result was found during attempts to precisely engineer the binding behavior of the conformational space of the DNA origami nanostructure. It was found that simple entropic chains, in the shape of single-stranded DNA strands, do not provide sufficient control over the conformation of the molecule. A combination with other interaction elements such as intermediate rigid double-stranded DNA strands as well as stacking interactions is required. The research on the validation of the binding behavior using these more complex building blocks was started in the course of the project, and is being continued in ongoing other research projects. The use of these building blocks for binding studies may also be evaluated for future patent applications in collaboration with the university technology transfer office, so that the nanodevice may contribute to future IP generation.
Concurrently with the described research work, literary research and discussions with experts in the field and key opinion leaders lead to the identification of proteins as the most promising field for targets for a potential functional nanodevice as well as for other DNA-origami nanostructure-based applications. Based on this, significant know-how on possible therapeutic targets, indications, manufacturers, researchers, and drugs in all stages of clinical development was established. This accumulation of information and expertise will be of great value for subsequent research projects on an even