MicroRNA Circuits for Transcriptional Logic

  • Madeleine Leisner
  • Leonidas Bleris
  • Jason Lohmueller
  • Zhen Xie
  • Yaakov BenensonEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 813)


One of the longstanding challenges in synthetic biology is rational design of complex regulatory circuitry with multiple biological inputs, complex internal processing, and physiologically active outputs. We have previously proposed how to address this challenge in the case of transcription factor inputs. Here we describe the methods used to construct these synthetic circuits, capable of performing logic integration of transcription factor inputs using microRNA expression vectors and RNA interference (RNAi). The circuits operate in mammalian cells and they can serve as starting point for more complex synthetic information processing networks in these cells.

Key words

Transcriptional regulation RNA interference Synthetic biology RNAi computing Mammalian cells Molecular logic 


  1. 1.
    Benenson, Y. (2009) Biocomputers: from test tubes to live cells, Molecular Biosystems 5, 675–685.PubMedCrossRefGoogle Scholar
  2. 2.
    Benenson, Y. (2009) RNA-based computation in live cells, Current Opinion in Biotechnology 20, 471–478.PubMedCrossRefGoogle Scholar
  3. 3.
    Weiss, R., Homsy, G. E., and Knight, T. F. (1999) Toward in vivo digital circuits, In Evolution as Computation: DIMACS Workshop (Landweber, L. F., and Winfree, E., Eds.), pp 275–295, Springer.Google Scholar
  4. 4.
    Gardner, T. S., Cantor, C. R., and Collins, J. J. (2000) Construction of a genetic toggle switch in Escherichia coli, Nature 403, 339–342.PubMedCrossRefGoogle Scholar
  5. 5.
    Elowitz, M. B., and Leibler, S. (2000) A synthetic oscillatory network of transcriptional regulators, Nature 403, 335–338.PubMedCrossRefGoogle Scholar
  6. 6.
    Isaacs, F. J., Dwyer, D. J., and Collins, J. J. (2006) RNA synthetic biology, Nature Biotechnology 24, 545–554.PubMedCrossRefGoogle Scholar
  7. 7.
    Canton, B., Labno, A., and Endy, D. (2008) Refinement and standardization of synthetic biological parts and devices, Nature Biotechnology 26, 787–793.PubMedCrossRefGoogle Scholar
  8. 8.
    Weber, W., and Fussenegger, M. (2002) Artificial mammalian gene regulation networks – novel approaches for gene therapy and bioengineering, Journal of Biotechnology 98, 161–187.PubMedCrossRefGoogle Scholar
  9. 9.
    Guet, C. C., Elowitz, M. B., Hsing, W. H., and Leibler, S. (2002) Combinatorial synthesis of genetic networks, Science 296, 1466–1470.PubMedCrossRefGoogle Scholar
  10. 10.
    Cox, R. S., Surette, M. G., and Elowitz, M. B. (2007) Programming gene expression with combinatorial promoters, Molecular Systems Biology 3, 11.Google Scholar
  11. 11.
    Ellis, T., Wang, X., and Collins, J. J. (2009) Diversity-based, model-guided construction of synthetic gene networks with predicted functions, Nature Biotechnology 27, 465–471.PubMedCrossRefGoogle Scholar
  12. 12.
    Kramer, B. P., Fischer, C., and Fussenegger, M. (2004) BioLogic gates enable logical transcription control in mammalian cells, Biotechnology and Bioengineering 87, 478–484.PubMedCrossRefGoogle Scholar
  13. 13.
    Rinaudo, K., Bleris, L., Maddamsetti, R., Subramanian, S., Weiss, R., and Benenson, Y. (2007) A universal RNAi-based logic evaluator that operates in mammalian cells, Nature Biotechnology 25, 795–801.PubMedCrossRefGoogle Scholar
  14. 14.
    Xie, Z., Liu, S. J., Bleris, L., and Benenson, Y. (2010) Logic integration of mRNA signals by an RNAi-based molecular computer, Nucleic Acids Research 38, 2692–2701.PubMedCrossRefGoogle Scholar
  15. 15.
    Leisner, M., Bleris, L., Lohmueller, J., Xie, Z., and Benenson, Y. (2010) Rationally designed logic integration of regulatory signals in mammalian cells, Nature Nanotechnology 5, 666–670.PubMedCrossRefGoogle Scholar
  16. 16.
    Shapiro, E., and Benenson, Y. (2006) Bringing DNA computers to life, Scientific American 294, 44–51.PubMedCrossRefGoogle Scholar
  17. 17.
    Baker, D., Group, B. F., Church, G., Collins, J., Endy, D., Jacobson, J., Keasling, J., Modrich, P., Smolke, C., and Weiss, R. (2006) Engineering life: Building a fab for biology, Scientific American 294, 44–51.Google Scholar
  18. 18.
    Benenson, Y. (2011) Engineering RNAi circuits, Methods in Enzymology 497, 187-205.PubMedGoogle Scholar
  19. 19.
    Chang, K., Elledge, S. J., and Hannon, G. J. (2006) Lessons from Nature: microRNA-based shRNA libraries, Nature Methods 3, 707–714.PubMedCrossRefGoogle Scholar
  20. 20.
    Stegmeier, F., Hu, G., Rickles, R. J., Hannon, G. J., and Elledge, S. J. (2005) A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells, Proceedings of the National Academy of Sciences of the United States of America 102, 13212–13217.PubMedCrossRefGoogle Scholar
  21. 21.
    Zhang, Z. L., Xie, Z., Zou, X. L., Casaretto, J., Ho, T. H. D., and Shen, Q. X. J. (2004) A rice WRKY gene encodes a transcriptional repressor of the gibberellin signaling pathway in aleurone cells, Plant Physiology 134, 1500–1513.PubMedCrossRefGoogle Scholar
  22. 22.
    Wagner, E. J., Baines, A., Albrecht, T., Brazas, R. M., and Garcia-Blanco, M. A. (2004) Imaging alternative splicing in living cells, Methods in Molecular Biology, 29–46.Google Scholar
  23. 23.
    Shu, X. K., Royant, A., Lin, M. Z., Aguilera, T. A., Lev-Ram, V., Steinbach, P. A., and Tsien, R. Y. (2009) Mammalian Expression of Infrared Fluorescent Proteins Engineered from a Bacterial Phytochrome, Science 324, 804–807.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Madeleine Leisner
    • 1
  • Leonidas Bleris
    • 2
  • Jason Lohmueller
    • 3
  • Zhen Xie
    • 4
  • Yaakov Benenson
    • 5
    Email author
  1. 1.Arnold Sommerfeld Centre for Theoretical PhysicsLudwigs-Maximilians-UniversityMunichGermany
  2. 2.Department of Bioengineering and Electrical EngineeringUniversity of Texas at DallasRichardsonUSA
  3. 3.Department of Biological and Biomedical SciencesHarvard Medical SchoolBostonUSA
  4. 4.Department of Biological EngineeringMassachusetts Institute of TechnologyCambridgeUSA
  5. 5.Department of Biosystems Science and EngineeringETH ZurichBaselSwitzerland

Personalised recommendations