Mammalian synthetic circuits with RNA binding proteins for RNA-only delivery

Journal name:
Nature Biotechnology
Year published:
Published online


Synthetic regulatory circuits encoded in RNA rather than DNA could provide a means to control cell behavior while avoiding potentially harmful genomic integration in therapeutic applications. We create post-transcriptional circuits using RNA-binding proteins, which can be wired in a plug-and-play fashion to create networks of higher complexity. We show that the circuits function in mammalian cells when encoded in modified mRNA or self-replicating RNA.

At a glance


  1. RNA-only multi-input microRNA classifier circuit differentiates between HeLa, HEK293 and MCF7 cells.
    Figure 1: RNA-only multi-input microRNA classifier circuit differentiates between HeLa, HEK293 and MCF7 cells.

    (a) An L7Ae-based multi-input microRNA classifier specifically recognizes HeLa cells based on a unique microRNA profile (highly expressed miR-21 and low levels of miR-141, miR-142(3p) and miR-146a). (b) Differential expression of output protein EGFP in HeLa, HEK293 and MCF7 cells with transient pDNA transfection. EGFP expression from the classifier circuit results in 18-fold and 25-fold higher output in HeLa cells in comparison to HEK293 and MCF7 cells, respectively (HEK293 fluorescence was normalized to 1, and circuit-regulated EGFP fluorescence was normalized to mKate (red fluorescent protein) expressed constitutively from the same promoter, to account for different expression levels across cell types). r.u., relative units. (c,d) Specific induction of apoptosis in HeLa cells by expression of circuit-controlled hBax protein compared with constitutive hBax expression: Annexin V positive cells in pDNA (c) and modRNA (d) transfected cells. (e) Cell death assay in a mixed HEK/HeLa-EBFP2 culture with modRNA delivery. The graphs indicate percent of dead cells as measured with AADvanced staining, with HEK293 and HeLa cells distinguished by EBFP2 fluorescence. EGFP-only transfection was used as a control in all apoptotic and cell death assays. Error bars represent s.d.

  2. Post-transcriptional cascades and two-state switch.
    Figure 2: Post-transcriptional cascades and two-state switch.

    (a) Cascade design for the pDNA and modRNA experiments. (b) Normalized mean EGFP fluorescence for the indicated cascade stages encoded either on pDNA or modRNA. Each stage n involves co-transfection of constructs 0 to n. (c) Replicon-encoded two-stage cascade. L7Ae was fused to mKate. Each replicon additionally encodes four nonstructural proteins (nsP1-4) and a subgenomic promoter (SGP) driving expression of circuit components. (d) Normalized mean EGFP and mKate fluorescence for cascade encoded in self-replicating RNA. (e) Switch design. Shaded: replicon components that include nsP1-4 and SGP. (f) Corresponding representative two-dimensional flow cytometry plots for pDNA and replicon transfections. (g,h) Normalized mean fluorescence of the two reporters in the different states of the switch encoded in pDNA (g) or replicon (h). Fluorescence was normalized to the lowest level in each chart. r.u., relative units; error bars represent s.d.


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Author information

  1. Present addresses: Global Biotherapeutics Technologies, Pfizer Inc., Cambridge, Massachusetts, USA (L.W.) and Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan (K.E.).

    • Liliana Wroblewska &
    • Kei Endo
  2. These authors contributed equally to this work.

    • Tasuku Kitada &
    • Kei Endo


  1. Department of Biological Engineering, Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Liliana Wroblewska,
    • Tasuku Kitada,
    • Velia Siciliano,
    • Breanna Stillo &
    • Ron Weiss
  2. Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.

    • Kei Endo &
    • Hirohide Saito
  3. The Hakubi Center for Advanced Research, Kyoto University, Kyoto, Japan.

    • Hirohide Saito


L.W. and R.W. conceived the ideas, L.W. designed and performed pDNA experiments, K.E. designed and performed modRNA experiments, T.K. designed and performed replicon experiments, V.S. designed and performed pDNA apoptotic assay and 3′ UTR repressor test in HeLa cells. B.S. created computational model of pDNA and replicon-based switch. L.W., R.W., K.E. and H.S. wrote the manuscript with input from all other authors.

Competing financial interests

H.S., K.E., R.W., L.W., T.K. and V.S. are co-inventors on patent applications covering the RNA circuits described here.

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Supplementary information

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    Supplementary Figures 1–28, Supplementary Tables 1–6 and Supplementary Notes 1–3

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  1. Supplementary Source Code (23 KB)

    RNA circuits model

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