Periodic Reporting for period 4 - AEDNA (Amorphous and Evolutionary DNA Nanotechnology)
Periodo di rendicontazione: 2020-12-01 al 2021-05-31
Problems addressed: Bottom-up synthetic biology aims at the construction of life-like systems from biomolecular components. In this context, DNA nanotechnology has been particularly successful in using DNA molecules to create molecular nanostructures of almost arbitrary shape, and to engineer biochemical reaction circuits that can sense, compute, control, or act. However, it is not clear whether and how DNA nanotechnology can be used to create larger, dynamical systems with life-like properties. The AEDNA project addressed two challenges associated with this issue. First, we aimed to explore ways to integrate dynamical DNA nanotechnology and other synthetic biology components derived from DNA nanotechnology into soft matter systems such as gels and emulsions. This allows to combine the nanoscale functionality of the DNA components with soft materials which can be patterned at much larger length scales, and which can be used to create artificial cell-like structures with embedded “DNA intelligence”. Second, we aimed to explore ways to utilize molecular evolution in the context of DNA and RNA nanotechnology. Even though DNA and RNA allow us “program” many of the properties of nanostructures and reaction circuits, some aspects are still difficult to design rationally. Utilization of evolutionary principles could potentially be used to improve DNA nanotechnological systems, and potentially let them evolve autonomously on the long run.
Relevance: Potential applications for DNA nanotechnology will require the integration of nanoscale components into macroscopic systems and materials. Intelligent, DNA functionalized gel materials developed in AEDNA will result in novel bioprinting possibilities, which can be useful for the realization of soft smart materials that can adapt to their environment and differentiate. This is important for the development of biomedical applications, and also for the emerging field of soft robotics. Evolutionary optimization of DNA structures, in particular DNA or RNA-scaffolded aptamers, can be used as novel biosensors and molecular binders with very high affinity, and could also be used as components for nanomedical robots. RNA circuits and switches developed in the project have applications in synthetic biology, where they could be used to improve diagnostic biocomputers or bioproduction processes.
In summary, the overall objectives of AEDNA were:
i) the development of a technology for multiscale self-organization based on DNA-functionalized gels which can be bioprinted to create artificial tissue-like materials. These materials will have basic information-processing and biochemical synthesis capabilities.
ii) the utilization of molecular evolution to develop novel or more powerful components for nucleic acid nanotechnology and synthetic biology.
Relevance: Potential applications for DNA nanotechnology will require the integration of nanoscale components into macroscopic systems and materials. Intelligent, DNA functionalized gel materials developed in AEDNA will result in novel bioprinting possibilities, which can be useful for the realization of soft smart materials that can adapt to their environment and differentiate. This is important for the development of biomedical applications, and also for the emerging field of soft robotics. Evolutionary optimization of DNA structures, in particular DNA or RNA-scaffolded aptamers, can be used as novel biosensors and molecular binders with very high affinity, and could also be used as components for nanomedical robots. RNA circuits and switches developed in the project have applications in synthetic biology, where they could be used to improve diagnostic biocomputers or bioproduction processes.
In summary, the overall objectives of AEDNA were:
i) the development of a technology for multiscale self-organization based on DNA-functionalized gels which can be bioprinted to create artificial tissue-like materials. These materials will have basic information-processing and biochemical synthesis capabilities.
ii) the utilization of molecular evolution to develop novel or more powerful components for nucleic acid nanotechnology and synthetic biology.
Within the AEDNA project, we developed protocols for the modification of gels with DNA molecules coding for various functions. We demonstrated that cell-free gene expression reactions could still proceed within these gels. From these modified gels, we created micrometer gel beads with different functionalities. We co-encapsulated multiple beads inside emulsion droplets, where they could cooperate and fulfil specific tasks, much like “organelles” in cells. We also created assemblies of droplet-based “artificial cells”, which communicated via small molecules, and utilized these small molecule signals to generate biochemical pulses or study simple forms of symmetry breaking.
In order to assemble the artificial cell-like structures more efficiently and on a larger scale, we developed methods for 3D printing of both DNA-functionalized gels and emulsion droplets. Within the 3D-printed gels, we could demonstrate simple forms of spatial differentiation, which was driven by diffusing DNA signals within the gels. In assemblies of emulsion droplets containing cell-free gene circuits, we were able to emulate spatial patterning based on the positional information contained in a morphogen gradient, similar as found in developmental processes in biology. We finally implemented microfluidic reactors to enable the execution of cell-free gene circuits with extended lifetimes, which allowed us to demonstrate period-doubling phenomena in a cell-free genetic oscillator.
AEDNA was also concerned with the use of DNA or RNA structures as scaffolds for aptamers – nucleic acid structures that, similar to antibodies, can bind to other molecules. We explored the influence of multivalent binding of target proteins to multiple DNA aptamers, and also the influence of flexibility, distances or orientations of the aptamers with respect to each other, resulting in a significant increase in target binding. We were also able to demonstrate the “selection” of best-binding DNA origami structures from a small library of structures with different aptamer arrangements.
Within AEDNA, we also established various RNA-based gene regulatory mechanisms (such as CRISPR interference and toehold riboregulators) to generate RNA-based gene circuits. We demonstrated various logic circuits based on toehold switches, and developed a novel approach to make CRISPR/Cas12a processes dependent on the presence of RNA input molecules, which was shown to work in the test tube, in bacteria and in human cells.
The project results were disseminated in 30 scientific publications, accompanied by published gene constructs and freely available software for 3D printing. Cell-free expression technology developed within AEDNA also was the basis for a very successful student team participating in the iGEM competition, which also led to the formation of the start-up company INVITRIS.
In order to assemble the artificial cell-like structures more efficiently and on a larger scale, we developed methods for 3D printing of both DNA-functionalized gels and emulsion droplets. Within the 3D-printed gels, we could demonstrate simple forms of spatial differentiation, which was driven by diffusing DNA signals within the gels. In assemblies of emulsion droplets containing cell-free gene circuits, we were able to emulate spatial patterning based on the positional information contained in a morphogen gradient, similar as found in developmental processes in biology. We finally implemented microfluidic reactors to enable the execution of cell-free gene circuits with extended lifetimes, which allowed us to demonstrate period-doubling phenomena in a cell-free genetic oscillator.
AEDNA was also concerned with the use of DNA or RNA structures as scaffolds for aptamers – nucleic acid structures that, similar to antibodies, can bind to other molecules. We explored the influence of multivalent binding of target proteins to multiple DNA aptamers, and also the influence of flexibility, distances or orientations of the aptamers with respect to each other, resulting in a significant increase in target binding. We were also able to demonstrate the “selection” of best-binding DNA origami structures from a small library of structures with different aptamer arrangements.
Within AEDNA, we also established various RNA-based gene regulatory mechanisms (such as CRISPR interference and toehold riboregulators) to generate RNA-based gene circuits. We demonstrated various logic circuits based on toehold switches, and developed a novel approach to make CRISPR/Cas12a processes dependent on the presence of RNA input molecules, which was shown to work in the test tube, in bacteria and in human cells.
The project results were disseminated in 30 scientific publications, accompanied by published gene constructs and freely available software for 3D printing. Cell-free expression technology developed within AEDNA also was the basis for a very successful student team participating in the iGEM competition, which also led to the formation of the start-up company INVITRIS.
The AEDNA project progressed beyond the state of the art in multiple ways:
- we have created DNA-functionalized gel materials which can be used for cell-free gene expression. Specifically, we have created several types of gel beads, which fulfil – depending on their modification – different functions, e.g. “sender” and “receiver” beads for molecular signals, or “activator” beads for switchable gene expression.
- we have combined dynamic DNA-nanotechnology and 3D bioprinting techniques for the first time.
- we have created small tissue-like arrangements of emulsion-based artificial cells that could communicate via small signals permeating through droplet interface bilayers and also membrane channels. With these, we demonstrated simple forms of spatial differentiation and also dynamical processes such as pulses of transcriptional activity running through such systems. We also explored the concept of “positional information” from developmental biology to synthetic bioprinted structures.
- we have realized period-doubling and quadrupling in a cell-free genetic oscillator, which had not been demonstrated before.
- we have created DNA origami-based scaffolds with several aptamer modifications, which considerably improve the binding of target molecules. In particular, we have systematically varied distance, orientation, and also linker flexibility, resulting in much higher binding yields than previously.
We further created RNA-based aptamer scaffolds that could be produced by in vitro transcription reactions.
- we realized the first switchable guide RNAs for CRISPR/Cas12a, opening up the possibility for conditional control of CRISPR processes.
- we have created DNA-functionalized gel materials which can be used for cell-free gene expression. Specifically, we have created several types of gel beads, which fulfil – depending on their modification – different functions, e.g. “sender” and “receiver” beads for molecular signals, or “activator” beads for switchable gene expression.
- we have combined dynamic DNA-nanotechnology and 3D bioprinting techniques for the first time.
- we have created small tissue-like arrangements of emulsion-based artificial cells that could communicate via small signals permeating through droplet interface bilayers and also membrane channels. With these, we demonstrated simple forms of spatial differentiation and also dynamical processes such as pulses of transcriptional activity running through such systems. We also explored the concept of “positional information” from developmental biology to synthetic bioprinted structures.
- we have realized period-doubling and quadrupling in a cell-free genetic oscillator, which had not been demonstrated before.
- we have created DNA origami-based scaffolds with several aptamer modifications, which considerably improve the binding of target molecules. In particular, we have systematically varied distance, orientation, and also linker flexibility, resulting in much higher binding yields than previously.
We further created RNA-based aptamer scaffolds that could be produced by in vitro transcription reactions.
- we realized the first switchable guide RNAs for CRISPR/Cas12a, opening up the possibility for conditional control of CRISPR processes.