Cells use spatial constraints to control and accelerate the flow of information in enzyme cascades and signalling networks. Synthetic silicon-based circuitry similarly relies on spatial constraints to process information. Here, we show that spatial organization can be a similarly powerful design principle for overcoming limitations of speed and modularity in engineered molecular circuits. We create logic gates and signal transmission lines by spatially arranging reactive DNA hairpins on a DNA origami. Signal propagation is demonstrated across transmission lines of different lengths and orientations and logic gates are modularly combined into circuits that establish the universality of our approach. Because reactions preferentially occur between neighbours, identical DNA hairpins can be reused across circuits. Co-localization of circuit elements decreases computation time from hours to minutes compared to circuits with diffusible components. Detailed computational models enable predictive circuit design. We anticipate our approach will motivate using spatial constraints for future molecular control circuit designs.
Strand displacement reactions are widely used in DNA nanotechnology as a building block for engineering molecular computers and machines. Here, we demonstrate that strand displacementbased probes can be triggered by RNA expressed in mammalian cells, thus taking a step towards adapting the DNA nanotechnology toolbox to a cellular environment. We systematically compare different probe architectures in order to identify a design that works robustly in living cells. Our optimized strand displacement probe combines chemically modified nucleic acids that enhance stability to degradation by cellular nucleases with structural elements that improve probe retention in the cytoplasm. We visualize probe binding to individual mRNA carrying 96 repeats of a target sequence in the 3'UTR. We find that RNA counts based on live cell imaging using a strand displacement probe are comparable to counts from independent measurement based on fluorescence in situ hybridization experiments. We used probes with scrambled toeholds and scrambled binding domains to demonstrate that target recognition indeed occurs through toeholdmediated strand displacement. Our results demonstrate that strand displacement probes can work reliably in mammalian cells and lay the groundwork for future applications of such probes for livecell imaging and molecular computing.
Cells use spatial constraints to control and accelerate the flow of information in enzyme cas-1 cades and signaling networks. Here we show that spatial organization can be a similarly into circuits that establish the universality of our approach. Because reactions preferen-7 tially occur between neighbors, identical DNA hairpins can be reused across circuits. Co-8 localization of circuit elements decreases computation time from hours to minutes compared 9 to circuits with diffusible components. Detailed computational models enable predictive cir-10 cuit design. We anticipate that our approach will motivate the use of spatial constraints in 11 molecular engineering more broadly, bringing embedded molecular control circuits closer to 12 1 . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/110965 doi: bioRxiv preprint first posted online Feb. 23, 2017; applications. 13Human-engineered systems, from ancient irrigation networks to modern semiconductor cir- architecture that exploits the advantages of spatial organization is still lacking. 53Here we experimentally demonstrate a modular design strategy -the "DNA domino" archi- The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/110965 doi: bioRxiv preprint first posted online Feb. 23, 2017; Localized signal propagation mechanism 61To illustrate how information is propagated spatially, we consider the "DNA domino effect" in a 62 minimal two-hairpin wire comprised of an Input and Output hairpin attached to a DNA origami 63 scaffold (Fig. 1b). In each reaction step, a hairpin stem is unwound and a toehold that is initially (Fig. 1c). We first confirmed that a signal could rapidly propagate across proxi-75 mally positioned Input and Output hairpins (single spacing) in a two-hairpin wire (t 1/2 <3 mins). 76No observable signal transfer was observed without input addition. We then doubled the distance 77 between Input and Output hairpins (double spacing) on the same origami, and showed that separat-78 ing the hairpins beyond their theoretical maximum reach resulted in minimal signal transfer (Fig. 79 1c, Supplementary Fig. S4). Furthermore, we found that interactions between Input and Output 80 hairpins on two different origamis were significantly slower than single-spaced hairpin interactions 81 on the same origami, and comparable to the double-spaced hairpin interactions. Crucially, decreas-82 ing the operating concentration of the origamis did not affect the speed of localized intra-origami 83 signal propagation, but significantly reduced the speed of non-localized inter-origami interactions 84 ( Supplementary Fig. S5). 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/110965 doi: bioRxiv preprint first posted online Feb. 23, 2017;We quantified the kinetics of domino circui...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.