Inspired by the remarkable ability of natural protein switches to sense and respond to a wide range of environmental queues, here we report a strategy to engineer synthetic protein switches by using DNA strand displacement to dynamically organize proteins with highly diverse and complex logic gate architectures. We show that DNA strand displacement can be used to dynamically control the spatial proximity and the corresponding fluorescence resonance energy transfer between two fluorescent proteins. Performing Boolean logic operations enabled the explicit control of protein proximity using multi-input, reversible and amplification architectures. We further demonstrate the power of this technology beyond sensing by achieving dynamic control of an enzyme cascade. Finally, we establish the utility of the approach as a synthetic computing platform that drives the dynamic reconstitution of a split enzyme for targeted prodrug activation based on the sensing of cancer-specific miRNAs.
Subscribe to Journal
Get full journal access for 1 year
only $14.08 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Govern, C. C. & ten Wolde, P. R. Optimal resource allocation in cellular sensing systems. Proc. Natl Acad. Sci. USA 111, 17486–17491 (2014).
Lalonde, S. et al. Molecular and cellular approaches for the detection of protein–protein interactions: latest techniques and current limitations. Plant J. 53, 610–635 (2008).
Papapostolou, D. & Howorka, S. Engineering and exploiting protein assemblies in synthetic biology. Mol. Biosyst. 5, 723 (2009).
Grünberg, R. & Serrano, L. Strategies for protein synthetic biology. Nucleic Acids Res. 38, 2663–2675 (2010).
Kelwick, R., MacDonald, J. T., Webb, A. J. & Freemont, P. Developments in the tools and methodologies of synthetic biology. Front. Bioeng. Biotechnol. 2, 60 (2014).
Sadowski, J. P., Calvert, C. R., Zhang, D. Y., Pierce, N. A. & Yin, P. Developmental self-assembly of a DNA tetrahedron. ACS Nano 8, 3251–3259 (2014).
Modi, S. et al. A DNA nanomachine that maps spatial and temporal pH changes inside living cells. Nat. Nanotechnol. 4, 325–330 (2009).
Krishnan, Y. & Simmel, F. C. Nucleic acid based molecular devices. Angew. Chem. Int. Ed. 50, 3124–3156 (2011).
Fu, J. et al. Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm. Nat. Nanotechnol. 9, 531–536 (2014).
Sun, Q., Madan, B., Tsai, S.-L., DeLisa, M. P. & Chen, W. Creation of artificial cellulosomes on DNA scaffolds by zinc finger protein-guided assembly for efficient cellulose hydrolysis. Chem. Comm. 50, 1423–1425 (2014).
Wilner, O. I. et al. Enzyme cascades activated on topologically programmed DNA scaffolds. Nat. Nanotechnol. 4, 249–254 (2009).
Piperberg, G., Wilner, O. I., Yehezkeli, O., Tel-Vered, R. & Willner, I. Control of bioelectrocatalytic transformations on DNA scaffolds. J. Am. Chem. Soc. 131, 8724–8725 (2009).
Zhang, D. Y. & Seelig, G. Dynamic DNA nanotechnology using strand-displacement reactions. Nat. Chem. 3, 103–113 (2011).
Seelig, G., Soloveichik, D., Zhang, D. Y. & Winfree, E. Enzyme-free nucleic acid logic circuits. Science 314, 1585–1588 (2006).
Dirks, R. M. & Pierce, N. A. Triggered amplification by hybridization chain reaction. Proc. Natl Acad. Sci. USA 101, 15275–15278 (2004).
Qian, L. & Winfree, E. Scaling up digital circuit computation with DNA strand displacement cascades. Science 332, 1196–1201 (2011).
Xin, L., Zhou, C., Yang, Z. & Liu, D. Regulation of an enzyme cascade reaction by a DNA machine. Small 9, 3088–3091 (2013).
Liu, M. et al. A DNA tweezer-actuated enzyme nanoreactor. Nat. Commun. 4, 2127 (2013).
Janssen, B. M. G., Engelen, W. & Merkx, M. DNA-directed control of enzyme-inhibitor complex formation: a modular approach to reversibly switch enzyme activity. ACS Synth. Biol. 4, 547–553 (2015).
Prokup, A. & Deiters, A. Interfacing synthetic DNA logic operations with protein outputs. Angew. Chem. Int. Ed. 53, 13192–13195 (2014).
Hwang, Y.-C., Chen, W. & Yates, M. V. Use of fluorescence resonance energy transfer for rapid detection of enteroviral infection in vivo. Appl. Environ. Microbiol. 72, 3710–3715 (2006).
Blackstock, D., Sun, Q. & Chen, W. Fluorescent protein-based molecular beacons by zinc finger protein-guided assembly. Biotechnol. Bioeng. 112, 236–241 (2015).
Cantera, J. L., Chen, W. & Yates, M. V. Detection of infective poliovirus by a simple, rapid, and sensitive flow cytometry method based on fluorescence resonance energy transfer technology. Appl. Environ. Microbiol. 76, 584–588 (2010).
Los, G. V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–382 (2008).
Blackstock, D. & Chen, W. Halo-tag mediated self-labeling of fluorescent proteins to molecular beacons for nucleic acid detection. Chem. Commun. 50, 13735–13738 (2014).
Kostal, J., Mulchandani, A., Gropp, K. E. & Chen, W. A temperature responsive biopolymer for mercury remediation. Environ. Sci. Technol. 37, 4457–4462 (2003).
Liu, F., Tsai, S. L., Madan, B. & Chen, W. Engineering a high-affinity scaffold for non-chromatographic protein purification via intein-mediated cleavage. Biotechnol. Bioeng. 109, 2829–2835 (2012).
Zhang, D. Y. & Winfree, E. Control of DNA strand displacement kinetics using toehold exchange. J. Am. Chem. Soc. 131, 17303–17314 (2009).
Chen, X., Briggs, N., McLain, J. R. & Ellington, A. D. Stacking nonenzymatic circuits for high signal gain. Proc. Natl. Acad. Sci. USA 110, 5386–5391 (2013).
Yin, P., Choi, H. M. T., Calvert, C. R. & Pierce, N. A. Programming biomolecular self-assembly pathways. Nature 451, 318–322 (2008).
Jiang, Y. S., Bhadra, S., Li, B. & Ellington, A. D. Mismatches improve the performance of strand-displacement nucleic acid circuits. Angew. Chem. Int. Ed. 53, 1845–1848 (2014).
Bandiera, S., Pfeffer, S., Baumert, T. F. & Zeisel, M. B. miR-122. A key factor and therapeutic target in liver disease. J. Hepatol. 62, 448–457 (2015).
Coulouarn, C., Factor, V. M., Andersen, J. B., Durkin, M. E. & Thorgeirsson, S. S. Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties. Oncogene 28, 3526–3536 (2009).
Agapakis, C. M., Boyle, P. M. & Silver, P. A. Natural strategies for the spatial optimization of metabolism in synthetic biology. Nat. Chem. Biol. 8, 527–535 (2012).
Dueber, J. E. et al. Synthetic protein scaffolds provide modular control over metabolic flux. Nat. Biotechnol. 27, 753–759 (2009).
Conrado, R. J., Varner, J. D. & DeLisa, M. P. Engineering the spatial organization of metabolic enzymes: mimicking nature’s synergy. Curr. Opin. Biotechnol. 19, 492–499 (2008).
Bayer, E. A., Belaich, J.-P., Shoham, Y. & Lamed, R. The cellulosomes: multienzyme machines for degradation of plant cell wall polysaccharides. Annu. Rev. Microbiol. 58, 521–554 (2004).
Sun, Q. & Chen, W. HaloTag mediated artificial cellulosome assembly on a rolling circle amplification DNA template for efficient cellulose hydrolysis. Chem. Commun. 52, 6701–6704 (2016).
Erbs, P. et al. In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene. Cancer Res. 60, 3813–3822 (2000).
Austin, E. A. & Huber, B. E. A first step in the development of gene therapy for colorectal carcinoma: cloning, sequencing, and expression of Escherichia coli cytosine deaminase. Mol. Pharmacol. 43, 380–387 (1993).
Ear, P. H. & Michnick, S. W. A general life–death selection strategy for dissecting protein functions. Nat. Methods 6, 813–816 (2009).
Hemphill, J. & Deiters, A. DNA computation in mammalian cells: microRNA logic operations. J. Am. Chem. Soc. 135, 10512–10518 (2013).
Groves, B. et al. Computing in mammalian cells with nucleic acid strand exchange. Nat. Nanotechnol. 11, 287–294 (2016).
Zhang, D. Y. & Winfree, E. Robustness and modularity properties of a non-covalent DNA catalytic reaction. Nucleic Acids Res. 38, 4182–4197 (2010).
Chang, J. et al. miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may downregulate the high affinity cationic amino acid transporter CAT-1. RNA Biol. 1, 106–113 (2004).
Laterza, O. F. et al. Plasma microRNAs as sensitive and specific biomarkers of tissue injury. Clin. Chem. 55, 1977–1983 (2009).
Xie, Z., Wroblewska, L., Prochazka, L., Weiss, R. & Benenson, Y. Multi-input RNAi-based logic circuit for identification of specific cancer cells. Science 333, 1307–1311 (2011).
Draghici, B. & Ilies, M. A. Synthetic nucleic acid delivery systems: present and perspectives. J. Med. Chem. 58, 4091–4130 (2015).
Lächelt, U. & Wagner, E. Nucleic acid therapeutics using polyplexes: a journey of 50 years (and beyond). Chem. Rev. 115, 11043–11078 (2015).
Lehto, T., Ezzat, K., Wood, M. J. A. & El Andaloussi, S. Peptides for nucleic acid delivery. Adv. Drug Deliv. Rev. 106, 172–182 (2016).
Fu, A., Tang, R., Hardie, J., Farkas, M. E. & Rotello, V. M. Promises and pitfalls of intracellular delivery of proteins. Bioconj. Chem. 25, 1602–1608 (2014).
Pisal, D. S., Kosloski, M. P. & Balu-Iyer, S. V. Delivery of therapeutic proteins. J. Pharm. Sci. 99, 2557–2575 (2010).
Tsai, S. L., Oh, J., Singh, S., Chen, R. & Chen, W. Functional assembly of mini-cellulosomes on the yeast surface for cellulose hydrolysis and ethanol production. Appl. Environ. Microbiol. 75, 6087–6093 (2009).
We thank S. Michnick for providing the split yCD constructs. This work was supported by grants from National Science Foundation (CBET1510817 and MCB1543838).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Chen, R.P., Blackstock, D., Sun, Q. et al. Dynamic protein assembly by programmable DNA strand displacement. Nature Chem 10, 474–481 (2018). https://doi.org/10.1038/s41557-018-0016-9
Current Opinion in Biotechnology (2020)
Binding Energy as Driving Force for Controllable Reconstruction of Hydrogen Bonds with Molecular Scissors
Journal of the American Chemical Society (2020)
Nanoparticles for Manipulation of the Developmental Wnt, Hedgehog, and Notch Signaling Pathways in Cancer
Annals of Biomedical Engineering (2020)
Point-of-care testing of protein biomarkers by integrating a personal glucose meter with a concatenated DNA amplifier
Sensors and Actuators B: Chemical (2020)