Our goal is the realization of self-organizing molecular and cellular systems that are able to respond to their environment, compute, move, take action. On the long term, we envision autonomous systems that are reconfigurable, that can evolve and develop.
Conditional guide RNAs for Cas12a in mammalian cells: Conditional guide RNAs (gRNAs) allow to make CRISPR-based processes such as gene editing or gene regulation dependent on cellular or environmental signals. We have developed a novel strategy to switch gRNAs for the CRISPR-associated protein Cas12a based on various molecular inputs - microRNAs, short hairpin RNAs, metabolites (via a ribozyme) or other RNA inputs (via a strand displacement process) in mammalian cells. Importantly, in this approach the guide RNAs are produced via a Pol II-promoter and later processed to become a fully functional gRNA. This also allows to encode a full gRNA circuit - including the mRNA encoding the Cas12a - on a single transcript.
L. Oesinghaus and F.C. Simmel, Controlling Gene Expression in Mammalian Cells Using Multiplexed Conditional Guide RNAs for Cas12a, Angew. Chem. Int. Ed. (2021). https://doi.org/10.1002/anie.202107258
Multi-aptamer scaffolds: Aptamers provide a “natural” interface between the DNA and protein world, and have been previously used to bind and arrange proteins on DNA nanostructures. Multivalent binding of target proteins by several aptamers is known to improve the binding strength, but the full potential of origami nanostructures to control the orientation of multiple binders, and also to adjust the flexibility of the binders to enhance their binding properties, has not been utilized so far. We now demonstrate a multiplexed single molecule assay based on DNA origami cavities to optimize the influence of geometry and molecular mechanical properties in two model protein-aptamer systems. Maybe most interestingly, our work also hints at the possibility to “evolve” DNA origami/aptamer scaffolds in a similar way as the SELEX method is used to evolve aptamers in the first place. As a first step in this direction, we demonstrate affinity selection of good binders from a mixture of origami structures.
A. Aghebat Rafat*, S. Sagredo*, M. Thalhammer & F. C. Simmel, Barcoded DNA origami structures for multiplexed optimization and enrichment of DNA-based protein-binding cavities, Nature Chemistry 12, 852-859 (2020). doi:10.1038/s41557-020-0504-6
3D printing meets dynamic DNA nanotechnology: Additive manufacturing enables the generation of 3D structures with defined shapes from a wide range of printable materials. Most of the materials employed so far are static and do not provide any intrinsic programma-bility or pattern-forming capability. On the other hand, the exquisite programmability of DNA-based systems has only rarely been used to generate functional materials on the mm-scale and above. In order to bring together “the best of both worlds”, we developed a low-cost 3D bioprinting approach using a DNA-functionalized bioink. We show that dynamic DNA nanotechnology can be used to control diffusion, addressable localization, and pattern formation in 3D printed gels.
J. Müller, A.C. Jäkel, D. Schwarz, L. Aufinger, F.C. Simmel, Programming Diffusion and Localization of DNA Signals in 3D-Printed DNA-Functionalized Hydrogels, Small 347, 2001815–10 (2020).
Multi-input logic for CRISPR-Cas12a: CRISPR mechanisms utilize the action of RNA-dependent nucleases to bind to and process DNA molecules whose sequence is determined by so-called guide RNAs (gRNAs), which make them natural candidates for regulation by RNA strand displacement processes. In this work, we utilized the intrinsic gRNA processing property of the CRISPR-associated protein Cas12a to implement guide RNAs that are switchable by RNA strand displacement reactions. Inactive strand-displacement gRNAs (SDgRNAs) can be activated by trigger RNA molecules, which induce a conformational change that facilitates binding of Cas12a. After processing of the SDgRNA, the resulting Cas12a-gRNA is fully active. Using this switchable principle, we demonstrate control of DNA processing by Cas12a in vitro via RNA-based input logic circuits. Using the catalytically inactive mutant dCas12a, we also realize SDgRNA-based logical control of gene expression in E.coli bacteria.
L. Oesinghaus, F. C. Simmel, Switching the activity of Cas12a using guide RNA strand displacement circuits, Nature Communications (2019). DOI: 10.1038/s41467-019-09953-w
Communicating artificial cells: Multicellularity in living organisms allows for complex behavior through differentiation of cell types. We have assembled artificial cells into synthetic "microtissues” through the use of water-in-oil droplets forming lipid bilayers upon contact with each other. We established and characterized cell-to-cell communication in these artificial multicellular structures. Using different types of synthetic in vitro gene circuits, we then implemented two examples of more complex dynamical behavior in such systems: spatial propagation of a chemical signal and a simple form of cellular differentiation.
A. Dupin, F. C. Simmel, Signalling and differentiation in emulsion-based multi-compartmentalized in vitro gene circuits. Nat Chem. 11, 32–39 (2019). https://www.nature.com/articles/s41557-018-0174-9
News and Views article by Li & Schulman: https://doi.org/10.1038/s41557-018-0192-7
Artificial gel based organelles: Our artificial organelles consist of agarose hydrogel beads functionalized with DNA molecules, which either act as genetic templates or which are able to capture diffusing RNA molecules. In order to mimic artificial cells, we encapsulated the organelles within emulsion droplets containing a cell-free gene expression system. One type of organelle was used to generate mRNA molecules coding for a fluorescent protein, whose translation was inhibited in the absence of certain trigger DNA molecules. Only when the RNA diffused into organelles containing the trigger, translation of the protein was activated. In this way, similar as in eukaryotic cells, we separated genetic transcription and translation processes into distinct subcellular compartments.
L. Aufinger, F. C. Simmel, Artificial Gel-based Organelles for Spatial Organization of Cell-free Gene Expression Reactions, Angew. Chem. Int. Ed. 57, 17245-17248 (2018). DOI: 10.1002/anie.201809374
Rise of the synthetic peptide cell: Compartmentalization and growth are central aspects of living cells, but the creation of natural membrane building blocks is difficult to implement in vitro. We now created peptide-based reaction compartments which are able to grow when the membrane peptide is expressed inside of them. By encapsulating a bacterial cell extract and DNA templates for protein expression we demonstrated the production of fluorescent proteins and amphiphilic elastin-like polypeptides in vesiculo which serve as the membrane building block.
K. Vogele, T. Frank, L. Gasser, M.A. Goetzfried, M. Hackl, S.A. Sieber, F.C. Simmel, T. Pirzer, Towards synthetic cells using peptide-based reaction compartments, Nature Communications 9: 3862 (2018). DOI: 10.1038/s41467-018-06379-8
DNA biochips: strand displacement lithography and gene expression. We present a biocompatible, DNA-based resist termed "Bephore" based on commercially available components, which can be patterned by both photo- and electron beam lithography. Bephore is well suited for multi-step lithographic processes, enabling the immobilization of different types of DNA molecules with micrometer precision, as shown in the miniaturized, fluorescent DNA-based replica of Franz Marc's painting "Tiger" (1912). As an application, we demonstrate compartmentalized, on-chip gene expression from three sequentially immobilized DNA templates, leading to spatially resolved protein expression gradients.
G. Pardatscher, M. Schwarz-Schilling, S. S. Daube, R. H. Bar-Ziv, and F. C. Simmel, Gene Expression on DNA Biochips Patterned with Strand Displacement Lithography. Angew. Chem. Int. Ed. 57, 4783-4786 (2018). DOI: 10.1002/anie.201800281
News and Views (Nature Chemistry): Link
Electrical control of a self-assembled DNA robot arm. We have created a nanoscale platform of size 55 nm x 55 nm with an integrated molecular arm that can rotate around a hinge in the center of the platform. In contrast to previous work on molecular machines based on DNA molecules, we utilized electrical fields to drive and control the motion of the arm with respect to the platform. With this technique, we can switch the position of the arm within milliseconds, which allows fast transport of molecules and nanoparticles. The robot arm can also be used to exert forces in the pico-Newton range, which is demonstrated in DNA unzipping experiments. Importanly, electrical manipulation allows us to realize complex, computer-controlled movement patterns.
E. Kopperger*, J. List*, S. Madhira, F. Rothfischer, D. C. Lamb, and F. C. Simmel, A self-assembled nanoscale robotic arm controlled by electric fields, Science 359, 296–301 (2018). DOI: 10.1126/science.aao4284