Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

A universal RNAi-based logic evaluator that operates in mammalian cells

Nature Biotechnology volume 25, pages 795–801 (2007)Cite this article

Abstract

Molecular automata1,2,3 that combine sensing4,5,6, computation7,8,9,10,11,12 and actuation13,14 enable programmable manipulation of biological systems. We use RNA interference (RNAi)15 in human kidney cells to construct a molecular computing core that implements general Boolean logic1,3,8,9,10,11,12,16 to make decisions based on endogenous molecular inputs. The state of an endogenous input is encoded by the presence or absence of 'mediator' small interfering RNAs (siRNAs). The encoding rules, combined with a specific arrangement of the siRNA targets in a synthetic gene network17, allow direct evaluation of any Boolean expression in standard forms using siRNAs and indirect evaluation using endogenous inputs. We demonstrate direct evaluation of expressions with up to five logic variables. Implementation of the encoding rules through sensory up- and down-regulatory links between the inputs and siRNA mediators will allow arbitrary Boolean decision-making using these inputs.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Design of the decision-making automaton that uses a DNF evaluator.
Figure 2: Design of the decision-making automaton that uses a CNF evaluator and automaton's input encoding rules.
Figure 3: Testing individual DNF clause molecules.

Similar content being viewed by others

References

  1. Stojanovic, M.N. & Stefanovic, D. A deoxyribozyme-based molecular automaton. Nat. Biotechnol. 21, 1069–1074 (2003).

    Article  CAS  Google Scholar 

  2. Benenson, Y., Gil, B., Ben-Dor, U., Adar, R. & Shapiro, E. An autonomous molecular computer for logical control of gene expression. Nature 429, 423–429 (2004).

    Article  CAS  Google Scholar 

  3. Macdonald, J. et al. Medium scale integration of molecular logic gates in an automaton. Nano Lett. 6, 2598–2603 (2006).

    Article  CAS  Google Scholar 

  4. Isaacs, F.J. et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat. Biotechnol. 22, 841–847 (2004).

    Article  CAS  Google Scholar 

  5. Bayer, T.S. & Smolke, C.D. Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat. Biotechnol. 23, 337–343 (2005).

    Article  CAS  Google Scholar 

  6. An, C.I., Trinh, V.B. & Yokobayashi, Y. Artificial control of gene expression in mammalian cells by modulating RNA interference through aptamer-small molecule interaction. RNA 12, 710–716 (2006).

    Article  CAS  Google Scholar 

  7. Ezziane, Z. DNA computing: applications and challenges. Nanotechnology 17, R27–R39 (2006).

    Article  CAS  Google Scholar 

  8. Basu, S. & Weiss, R. The device physics of cellular logic gates. First Workshop on Non-Silicon Computation Boston, February 3 2002 <http://www.cs.cmu.edu/∼phoenix/nsc1/paper/3-2.pdf> 54–61.

    Google Scholar 

  9. Balzani, V., Credi, A. & Venturi, M. Molecular logic circuits. ChemPhysChem 4, 49–59 (2003).

    Article  CAS  Google Scholar 

  10. Kramer, B.P., Fischer, C. & Fussenegger, M. BioLogic gates enable logical transcription control in mammalian cells. Biotechnol. Bioeng. 87, 478–484 (2004).

    Article  CAS  Google Scholar 

  11. Baron, R., Lioubashevski, O., Katz, E., Niazov, T. & Willner, I. Elementary arithmetic operations by enzymes: A model for metabolic pathway based computing. Angew. Chem. Int. Ed. 45, 1572–1576 (2006).

    Article  CAS  Google Scholar 

  12. Seelig, G., Soloveichik, D., Zhang, D.Y. & Winfree, E. Enzyme-free nucleic acid logic circuits. Science 314, 1585–1588 (2006).

    Article  CAS  Google Scholar 

  13. Kolpashchikov, D.M. & Stojanovic, M.N. Boolean control of aptamer binding sites. J. Am. Chem. Soc. 127, 11348–11351 (2005).

    Article  CAS  Google Scholar 

  14. Beyer, S. & Simmel, F.C. A modular DNA signal translator for the controlled release of a protein by an aptamer. Nucleic Acids Res. 34, 1581–1587 (2006).

    Article  CAS  Google Scholar 

  15. Eckstein, F. Small non-coding RNAs as magic bullets. Trends Biochem. Sci. 30, 445–452 (2005).

    Article  CAS  Google Scholar 

  16. Penchovsky, R. & Breaker, R.R. Computational design and experimental validation of oligonucleotide-sensing allosteric ribozymes. Nat. Biotechnol. 23, 1424–1433 (2005).

    Article  CAS  Google Scholar 

  17. Hasty, J., McMillen, D. & Collins, J.J. Engineered gene circuits. Nature 420, 224–230 (2002).

    Article  CAS  Google Scholar 

  18. Wiener, N. Cybernetics (MIT Press, New York, 1948).

    Google Scholar 

  19. Soloveichik, D. & Winfree, E. The computational power of Benenson automata. Theor. Comp. Sci. 244, 279–297 (2005).

    Article  Google Scholar 

  20. Weiss, R., Homsy, G. & Knight, T.F. Toward in vivo digital circuits. in Natural Computing Series (eds. Winfree, E. Landweber, L.F.) 275–295, (Springer, Heidelberg, 2002).

    Google Scholar 

  21. Buchler, N.E., Gerland, U. & Hwa, T. On schemes of combinatorial transcription logic. Proc. Natl. Acad. Sci. USA 100, 5136–5141 (2003).

    Article  CAS  Google Scholar 

  22. Isaacs, F.J., Dwyer, D.J. & Collins, J.J. RNA synthetic biology. Nat. Biotechnol. 24, 545–554 (2006).

    Article  CAS  Google Scholar 

  23. Laslo, P. et al. Multilineage transcriptional priming and determination of alternate hematopoietic cell fates. Cell 126, 755–766 (2006).

    Article  CAS  Google Scholar 

  24. Rosenwald, A. et al. Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia. J. Exp. Med. 194, 1639–1647 (2001).

    Article  CAS  Google Scholar 

  25. Doench, J.G. & Sharp, P.A. Specificity of microRNA target selection in translational repression. Genes Dev. 18, 504–511 (2004).

    Article  CAS  Google Scholar 

  26. Schwarz, D.S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003).

    Article  CAS  Google Scholar 

  27. Caron, L. et al. The Lac repressor provides a reversible gene expression system in undifferentiated and differentiated embryonic stem cell. Cell. Mol. Life Sci. 62, 1605–1612 (2005).

    Article  CAS  Google Scholar 

  28. Sullivan, C.S. & Ganem, D. A virus-encoded inhibitor that blocks RNA interference in mammalian cells. J. Virol. 79, 7371–7379 (2005).

    Article  CAS  Google Scholar 

  29. Yokobayashi, Y., Weiss, R. & Arnold, F.H. Directed evolution of a genetic circuit. Proc. Natl. Acad. Sci. USA 99, 16587–16591 (2002).

    Article  CAS  Google Scholar 

  30. Hooshangi, S., Thiberge, S. & Weiss, R. Ultrasensitivity and noise propagation in a synthetic transcriptional cascade. Proc. Natl. Acad. Sci. USA 102, 3581–3586 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank A. Murray, E. O'Shea, G. Church, D. Weitz, X. Xie, T. Hwa, C. Queitsch, A. Rivzi, G. Kudla and K. Foster for discussions; I. Benenson for the critical review of the manuscript; anonymous reviewers for insightful comments; and K. Thorn and B. Tilton for technical support. The EF1-α promoter was amplified by PCR from pLEIGW (a gift from Ihor Lemischka, Princeton University). The plasmid pCAGOP containing the synthetic chicken β-actin promoter with lac operators (CAGOP) was obtained from B. Binétruy27. pLV-tTRKRAB-Red was a gift of D. Trono, Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland. The work was supported by the Bauer Fellows program and by the National Institute of General Medical Sciences (NIGMS) grant 5P50 GM068763-01. K.R. was partially supported by Harvard's Program for Research in Science and Engineering (PRISE).

Author information

Author notes

  1. Keller Rinaudo and Leonidas Bleris: These authors contributed equally to this work.

Authors and Affiliations

  1. FAS Center for Systems Biology, Harvard University, 7 Divinity Ave., Cambridge, 02138, Massachusetts, USA

    Keller Rinaudo, Leonidas Bleris, Rohan Maddamsetti & Yaakov Benenson

  2. Department of Electrical Engineering, Princeton University, E-Quad B-312, Olden St., Princeton, 08544, New Jersey, USA

    Sairam Subramanian & Ron Weiss

  3. Department of Molecular Biology, Princeton University, E-Quad B-312, Olden St., Princeton, 08544, New Jersey, USA

    Sairam Subramanian & Ron Weiss

Authors
  1. Keller Rinaudo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Leonidas Bleris
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rohan Maddamsetti
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sairam Subramanian
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ron Weiss
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yaakov Benenson
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.B. and R.W. designed research; Y.B., K.R., L.B., R.M. and S.S. performed research; Y.B., R.W., K.R. and L.B. wrote the paper.

Corresponding authors

Correspondence to Ron Weiss or Yaakov Benenson.

Ethics declarations

Competing interests

There is a pending patent application that covers the results presented in this paper.

Supplementary information

Supplementary Fig. 1

Basic notions of the Boolean logic. (PDF 96 kb)

Supplementary Fig. 2

Crosstalk verification between siRNAs and their targets. (PDF 150 kb)

Supplementary Fig. 3

Influence of the secondary structure on siRNA efficiency. (PDF 142 kb)

Supplementary Fig. 4

The histogram of the expression levels. (PDF 63 kb)

Supplementary Fig. 5

Alternative sensory module designs. (PDF 54 kb)

Supplementary Table 1

Association between the literals and the siRNAs. (PDF 34 kb)

Supplementary Table 2

Sequences of the oligonucleotides employed in the circuit construction. (PDF 69 kb)

Supplementary Methods (PDF 57 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rinaudo, K., Bleris, L., Maddamsetti, R. et al. A universal RNAi-based logic evaluator that operates in mammalian cells. Nat Biotechnol 25, 795–801 (2007). https://doi.org/10.1038/nbt1307

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1307

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing