De novo automated design of small RNA circuits for engineering synthetic riboregulation in living cells

Abstract

A grand challenge in synthetic biology is to use our current knowledge of RNA science to perform the automatic engineering of completely synthetic sequences encoding functional RNAs in living cells. We report here a fully automated design methodology and experimental validation of synthetic RNA interaction circuits working in a cellular environment. The computational algorithm, based on a physicochemical model, produces novel RNA sequences by exploring the space of possible sequences compatible with predefined structures. We tested our methodology in Escherichia coli by designing several positive riboregulators with diverse structures and interaction models, suggesting that only the energy of formation and the activation energy (free energy barrier to overcome for initiating the hybridization reaction) are sufficient criteria to engineer RNA interaction and regulation in bacteria. The designed sequences exhibit nonsignificant similarity to any known noncoding RNA sequence. Our riboregulatory devices work independently and in combination with transcription regulation to create complex logic circuits. Our results demonstrate that a computational methodology based on first-principles can be used to engineer interacting RNAs with allosteric behavior in living cells.

Fig. 1.

Schemes of methodology and designs. (A) Thermodynamic scheme of RNA interaction, showing the different free energies at play and the progression of the reaction. We define the reaction coordinate as the size of intermolecular pairs (d). (B) Optimization scheme followed to design the RNA devices. (C) Secondary structures specified for the single species to obtain different RNA devices. Nucleotides shown were maintained fixed; RBS sequence yellow colored. Different devices were designed by imposing different structures for the riboregulator.

Fig. 2.

Schematic representation of the six different RNA devices we designed and engineered for riboregulation. Devices RAJ11 and RAJ12 were obtained by imposing the structure T4, device RAJ21 with T1, device RAJ22 with T2, device RAJ23 with T3, and device RAJ31 with T5. SI Appendix (Fig. S7) shows the helical structure of the different complexes together with the corresponding base-pairing probability matrixes. SI Appendix (Table S1) shows the sequences of species. SI Appendix (Table S4) shows the thermodynamic properties of the systems.

Fig. 3.

Experimental characterization of the RNA devices. Normalized fluorescence of the devices together with the apparent activation fold (Top), measured as the ratio of fluorescence in presence and absence of the riboregulator (see also SI Appendix, Fig. S15, for flow cytometry characterizations).

Fig. 4.

Context analysis of the RNA devices. (A) Comparison of the activity of the device RAJ11 in the regular strain (JS006) and in a strain ΔRNase III (HT115). (B) Orthogonality analysis of the devices, showing the fraction of complex formation at the equilibrium (values provided in SI Appendix, Table S5; see also SI Appendix, Fig. S11). (C) Experimental validation of the orthogonality between the devices RAJ11 and RAJ12.

Fig. 5.

The RNA device can work in combination with transcription regulation. (A) Scheme of a circuit coupling these two types of control (resulting in an AND logic gate), where IPTG and aTc are the two inputs and GFP the output. To implement this circuit we used the device RAJ11. (B) Experimental characterization of the circuit showing the dynamics of the normalized fluorescence after introduction of inducers at time 0 (see also SI Appendix, Fig. S19, for the full set of dynamics at different levels of IPTG and aTc). (C) Arbitrary units of GFP (colored circles) are estimated by taking the maximum value of the dynamics shown in SI Appendix (Fig. S19), ensuring OD₆₀₀ < 0.4, and dividing this value by the one obtained in case of no inducers in the medium. Solid lines are obtained with the mathematical model presented in SI Appendix (Eq. S7), with parameters fitted against these experimental data.