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.
