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A general design strategy for protein-responsive riboswitches in mammalian cells

Nature Methods volume 11, pages 1154–1160 (2014)Cite this article

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Abstract

RNAs are ideal for the design of gene switches that can monitor and program cellular behavior because of their high modularity and predictable structure-function relationship. We have assembled an expression platform with an embedded modular ribozyme scaffold that correlates self-cleavage activity of designer ribozymes with transgene translation in bacteria and mammalian cells. A design approach devised to screen ribozyme libraries in bacteria and validate variants with functional tertiary stem-loop structures in mammalian cells resulted in a designer ribozyme with a protein-binding nutR-boxB stem II and a selected matching stem I. In a mammalian expression context, this designer ribozyme exhibited dose-dependent translation control by the N-peptide, had rapid induction kinetics and could be combined with classic small molecule–responsive transcription control modalities to construct complex, programmable genetic circuits.

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Figure 1: Expression platform with embedded modular ribozyme scaffold.
Figure 2: Validation of self-cleavage performance of HHRs with nutR-matching structural elements.
Figure 3: Bacterial cell–based strategy to select nutR-boxB–containing HHR variants with loop II sequences restoring the tertiary interloop structures.
Figure 4: Gene-switch performance of the designer ribozyme Env140-nutR-H6 in mammalian cells.
Figure 5: A three-input AND logic gate combining translation and transcriptional gene expression control.

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Acknowledgements

We thank P. Silver (Harvard Medical School) for providing the U1A-encoding vector pPS2042; M. Müller, F. Sedlmayer, M. Wieland, A. Bosshart and M. Jeschek for generous advice; and S. Stebler for experimental assistance. We are grateful to the single cell unit team V. Jäggin, M. Dessing and T. Horn for assistance with flow cytometry and fluorescence microscopy. This work was supported by a European Research Council (ERC) advanced grant (no. 321381) and in part by the Cantons of Basel and the Swiss Confederation within the INTERREG IV A.20 tri-national research program.

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Authors and Affiliations

  1. Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland

    Simon Ausländer, Pascal Stücheli, David Ausländer & Martin Fussenegger

  2. Department of Chemistry, University of Konstanz, Konstanz, Germany

    Charlotte Rehm & Jörg S Hartig

  3. Faculty of Science, University of Basel, Basel, Switzerland

    Martin Fussenegger

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  1. Simon Ausländer
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Contributions

S.A. designed the project. S.A., P.S., C.R., D.A., J.S.H. and M.F. analyzed results and wrote the manuscript. S.A., P.S., C.R. and D.A. performed the experimental work.

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Correspondence to Martin Fussenegger.

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Integrated supplementary information

Supplementary Figure 1 Design of the HHR expression platform.

PmeI/SpeI restriction endonucleases are used for simple one-step swapping of the HHR scaffold among bacterial and mammalian expression vectors. Whereas the bacterial expression vector contains a PT7-driven eGFP expression unit (pET16b-eGFP) with the HHR scaffold embedded in the 5’ untranslated region, the mammalian expression vector harbors a PSV40-driven SEAP expression unit (pSEAP2-control) with the HHR scaffold inserted in the 3’ untranslated region.

Supplementary Figure 2 Ribozyme-dependent EGFP expression profiles in HEK-293 cells.

GFP expression profiles correlating with the self-cleavage performance of Env25ac/inac (pDA230/pDA231), Env140ac/inac (pDA228/pDA229) and Env143ac/inac (pDA232/pDA233) HHRs in HEK-293 cells (set-up shown in Fig. 1c). Error bars are mean±SD of a representative experiment performed in triplicates.

Supplementary Figure 3 Detailed experimental guidelines for the selection of designer ribozymes.

The HHR scaffold expression library containing degenerated nucleotides in the desired loop structure is constructed by site-directed PCR-based mutagenesis using the bacterial expression vector as template. The HHR expression library is transformed into bacteria and functional hybrid ribozymes correlating with high-level eGFP expression are enriched by FACS-mediated sorting. The library subpopulation containing HHR variants with highest self-cleavage performance in bacteria is swapped into the corresponding mammalian expression vector and transformed again into bacteria to isolate clonal HHR variants which are subsequently transfected into mammalian cells for validation. The best-in-class designer ribozymes with optimal functional tertiary inter-loop contacts were identified in mammalian cells by correlating low-level SEAP expression and characterized by sequence analysis.

Supplementary Figure 4 General strategy for the design of protein-responsive mammalian riboswitches.

(a) Ribozyme candidates with high structural homology to a desired natural ribonucleoprotein (RNP) complex are identified in a comprehensive database of natural HHR sequences. (b) Replacement of the natural ribozyme’s stem loop by the RNP-derived stem loop creates a hybrid ribozyme whose self-cleavage performance is scored in a bacterial and mammalian expression context. (c) A complementary stem loop structure matching the RNP-derived stem loop to form functional inter-loop contacts is selected from a library of the hybrid ribozyme containing degenerated loop sequences by FACS-mediated selection in bacteria and validation in mammalian cells using a cross-kingdom ribozyme expression platform. The selected designer ribozyme enables protein-adjustable translation control in mammalian cells.

Supplementary Figure 5 Ribozyme performance of selected Env140-nutR-5Nac HHR clones in HEK-293 cells.

SEAP expression profiles correlating with the self-cleavage performance of selected Env140/nutR/5Nac HHR clones in HEK-293 cells relative to active Env140ac (100%) and inactive Env140inac (0%). The blue bar represents the parental hybrid ribozyme Env140/nutRac, which served as a template for the degenerated loop I library.

Supplementary Figure 6 Schematic representation of N-peptide–triggered Env140-nutR-H6–mediated SEAP expression control in mammalian cells.

In the absence of N-peptide self-cleavage of the designer ribozyme Env140/nutR/H6 embedded in the 3’ untranslated region of a SEAP-encoding mRNA (pPST67[H6]) removes the poly(A) tail which leads to nuclease (red pacman) –mediated destruction and shut down of SEAP expression. However, binding of N-peptide (pSA776) to stem loop II of Env140/nutR/H6 prevents formation of the tertiary inter-loop structures which inactivates the designer ribozyme and SEAP is translated from an intact mRNA exported to the cytosol and efficiently secreted by the host cell.

Supplementary Figure 7 Fluorescence micrographs of mCherry-transfected and N-peptide–mCherry–transfected mammalian cells.

Fluorescence micrographs of different cell lines containing the PSV40-driven expression mCherry expression vector (pFS29) or a PSV40-driven expression unit encoding the N-peptide-mCherry fusion protein (pSA776). Micrographs show the bright field (BF) channel as well as the 561nm-laser channel (561nm). Micrographs were taken 48 hours after transfection.

Supplementary Figure 8 Impact of N-peptide on the performance of different active and isogenic inactive HHR constructs.

(a-b) Impact of N-peptide on the performance of different active (a) and isogenic inactive (b) HHR constructs and the corresponding SEAP expression levels. Black bars: mCherry (pFS29); blue bars: N-peptide-mCherry (pSA776). SEAP expression analysis was performed 22 hours after transfection. Ac., active; inac., inactive. Error bars are mean±SEM (n=3). Please see Supplementary Table 1 for plasmid design and Supplementary Table 2 for ribozyme sequence.

Supplementary Figure 9 SEAP expression kinetics of active Env140ac and inactive Env140-nutR-H6inac ribozymes in CHO-K1 cells.

(a) SEAP expression kinetics of CHO-K1 cells cotransfected with active Env140ac HHR-embedded SEAP expression vector (pSA741) and either the mCherry- (pFS29; black line) or the N-peptide-mCherry-encoding expression vector (pSA776; blue line). (b) SEAP expression kinetics of CHO-K1 cells cotransfected with inactive Env140/nutR/H6inac HHR-embedded SEAP expression vector (pPST69) and either mCherry- (pFS29; black line) or the N-peptide-mCherry encoding expression vector (pSA776; blue line). Ac., active; inac., inactive. Error bars are mean±SEM (n=3).

Supplementary Figure 10 In vitro cleavage kinetics of the Env140ac ribozyme.

In-vitro cleavage kinetics of the Env140ac ribozyme in the presence (blue squares) and absence (black circles) of 4μM N-peptide. The cleavage kinetics of the inactive Env140inac ribozyme is shown in red diamonds.

Supplementary Figure 11 Impact of N-peptidemut/nat on Env140ac– and Env140-nutR-H6ac–controlled EGFP expression in bacterial cells.

(a) Amino acid identity of native and mutated N-peptide (N-peptidenat/mut). (b) Flow cytometry-based histograms showing eGFP-mediated fluorescence correlating with the self-cleavage performance of Env140ac (pPST49) or Env140/nutR/H6ac (pPST106) in the presence of native (pPST102) or mutated (pPST171) N-peptide in E. coli. Red peak indicates co-transformation with native N-peptide, blue peak with mutated N-peptide.

Supplementary Figure 12 Engineering a U1Ap-responsive designer ribozyme.

(a) Secondary structure of the U1hpII RNA motif. (b) Secondary RNA structures of stem loops I & II of the Env14 hammerhead ribozyme embedded on the ribozyme scaffold. (c) Flow cytometry-based histograms showing eGFP-mediated fluorescence correlating with the self-cleavage performance of Env14ac/inac (pPST172/pPST173) HHRs in bacteria (set-up shown in Fig. 1b). Red peaks indicate active HHR, blue peaks show inactive HHR and the dashed peak represents the autofluorescence of bacteria. (d) SEAP expression profiles correlating with the self-cleavage performance of Env14ac/inac (pSA909/pSA917) in CHO-K1 cells (set-up shown in Fig. 1c). (e) SEAP-based performance analysis of Env14ac stem loop I variants in CHO-K1 cells. Nucleotide mutations compared to the wild-type stem loop I are shown in blue. The ribozyme activity was normalized to the cleavage activity of the active (100%) and inactive Env14 (0%). (f) Profiling of the U1Ap-triggered SEAP expression in CHO-K1 cells co-transfected with the SEAP expression vector containing a 3’-UTR-embedded Env14ac (pSA909), Env14/A4Gac (pSA912), Env14/CGac (pSA914) Env14/U1hpIIac (pSA928) or Env140/nutR/H6ac (pPST67[H6]) and either mCherry (pFS20; black bars) or U1Ap-mCherry (pSA906; orange bars). The results for Env14/U1hpIIac (pSA928) and Env140/nutR/H6ac (pPST67[H6]) are also shown in Figure 4j and are here included for comparison. eGFP/SEAP expression was analyzed 22 hours after inoculation/transfection. Ac., active; inac., inactive. Error bars are mean±SEM (n=3).

Supplementary Figure 13 Two-input logic gates combining ribozyme-based N-peptide–triggered translation control with transcription switches.

(a) Genetic switchboard of the AND Boolean logic gate circuit consisting of the doxycycline-inducible Ptight-driven N-peptide-mCherry expression unit (pSA781) transactivated by rtTA (reverse tetracycline-dependent transactivator; pTet-ON) and a vanillic acid-inducible PVANON8-driven SEAP expression unit with 3’-UTR-embedded HHR (Env140/nutR/H6ac; pSA783) whose transcription is transrepressed by VanA4 (vanillic acid-dependent transrepressor; pCK188) and translation is modulated by N-peptide. (b) Combining the two input signals according to the truth table programs transfected HEK-293 cells to produce the enzymatic output SEAP only in the presence of both input signals doxycyline (Dox) and vanillic acid (Van). (c) Genetic switchboard of the B N-IMPLY A Boolean logic gate circuit consisting of an erythromycin-repressible PETR2-driven N-peptide-mCherry expression unit (pSA763) transactivated by ET1 (erythromycin-dependent transactivator; pWW35) a vanillic acid-inducible PVANON8-driven SEAP expression unit with 3’-UTR-embedded HHR (Env140/nutR/H6ac; pSA783) whose transcription is transrepressed by VanA4 (vanillic acid-dependent transrepressor; pCK188) and translation is modulated by N-peptide. (d) Combining the two input according to the truth table programs transfected HEK-293 cells to produce the enzymatic output SEAP only in the presence of the vanillic acid (Van) input and not in the presence of erythromycin (Ery).

Supplementary Figure 14 Dose dependence of the small-molecule–responsive transcription-control systems used for the assembly of logic gates.

(a) HEK-293 cells cotransfected with the vanillic acid-dependent transrepressor (VanA4; pCK188) and the VanA4-specific PvanON8-driven SEAP expression vector with embedded Env140/nutR/H6ac (PVanON8-SEAP-pA; pSA783) and either N-peptide-mCherry (pSA776; circles) or mCherry (pFS29; squares) were programmed with different concentrations of vanillic acid and profiled for SEAP expression after 40h. (b) HEK-293 cells were cotransfected with the reverse tetracycline-dependent transactivator (rtTA; pTet-ON) and the rtTA-specific Ptight-driven N-peptide-mCherry expression vector (Ptight-N-peptide-mCherry-pA; pSA781) and programmed with different concentrations of the antibiotic doxycyline. Red fluorescence was quantified by flow cytometry 40 hours after transfection. (c) HEK-293 cells cotransfected with the erythromycin-dependent transactivator (ET1; pWW35) and the ET1-specific PETR2-driven driven N-peptide-mCherry expression vector (PETR2-N-peptide-mCherry-pA; pSA763) and programmed with different concentrations of the antibiotic erythromycin. Red fluorescence was quantified using flow cytometry 40 hours after transfection. Error bars are mean±SEM (n=3).

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Ausländer, S., Stücheli, P., Rehm, C. et al. A general design strategy for protein-responsive riboswitches in mammalian cells. Nat Methods 11, 1154–1160 (2014). https://doi.org/10.1038/nmeth.3136

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