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