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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - AEDNA (Amorphous and Evolutionary DNA Nanotechnology)

Teaser

Summary

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 act as molecular detectors, computers, or control systems. 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 addresses two challenges associated with this issue. First, we 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 will allow us 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 â€œDNA intelligenceâ€. Second, we explore ways to utilize molecular evolution in the context of DNA and RNA nanotechnology. Even though DNA and RNA allow us â€œprogramâ€ many of the properties of nanostructures and reaction circuits, some aspects are still very difficult to design rationally. It is therefore tempting to utilize evolutionary principles to improve DNA nanotechnological systems, and potentially let them evolve autonomously.

Relevance for society:
Only if we manage to bridge the scales from nanoscopic to macroscopic, we can expect genuine applications arising from DNA nanotechnology. Intelligent, DNA functionalized gel materials developed in AEDNA will result in novel bioprinting possibilities, which could be useful for the realization of soft smart materials, which 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 help to create 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.

Overall objectives of AEDNA:
In summary, the overall objectives of AEDNA are:
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.

Work performed

Within the AEDNA project, we developed novel protocols for the modification of gels with DNA coding for various functions. Gene expression reactions could still proceed within these gels. From these modified gels, we created small gel beads with different functionalities. We co-encapsulated multiple beads inside emulsion droplets, where they could interact with each other and fulfil specific tasks, much like â€œorganellesâ€ in cells. We also created small assemblies of droplet-based artificial cells, which communicated via small molecules rather than nucleic acid signals. We 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 gel beads and emulsion droplets.

AEDNA was also concerned with the use of DNA or RNA structures as scaffolds for aptamers â€“ 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. AEDNA further aimed at the implementation of evolutionary techniques for the improvement of nucleic acid structures and dynamical systems. In this context, we established various RNA-based gene regulatory mechanisms (such as CRISPR interference and toehold riboregulators) to generate RNA-based cell-free gene circuits, which will be optimized by directed evolution in the next phase of the project.

Final results

We have already progressed beyond the state of the art in several 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 â€“ depending on their modification â€“ different functions, e.g., â€œsenderâ€ and â€œreceiverâ€ beads for molecular signals, or â€œactivatorâ€ beads for switchable gene expression.
- 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 have created nuecleic acid 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.

Until the end of the project, we anticipate further progress by utilizing our gels for 3D bioprinting, and also by demonstrating directed evolution of novel functions for nucleic acid nanotechnology.