Evaluating riboswitch optimality

Abstract

Riboswitches are RNA elements that recognize diverse chemical and biomolecular inputs, and transduce this recognition process to genetic, fluorescent, and other engineered outputs using RNA conformational changes. These systems are pervasive in cellular biology and are a promising biotechnology with applications in genetic regulation and biosensing. Here, we derive a simple expression bounding the activation ratio-the proportion of RNA in the active vs. inactive states-for both ON and OFF riboswitches that operate near thermodynamic equilibrium: 1+[I]/K_d^I, where [I] is the input ligand concentration and K_d^I is the intrinsic dissociation constant of the aptamer module toward the input ligand. A survey of published studies of natural and synthetic riboswitches confirms that the vast majority of empirically measured activation ratios have remained well below this thermodynamic limit. A few natural and synthetic riboswitches achieve activation ratios close to the limit, and these molecules highlight important principles for achieving high riboswitch performance. For several applications, including "light-up" fluorescent sensors and chemically-controlled CRISPR/Cas complexes, the thermodynamic limit has not yet been achieved, suggesting that current tools are operating at suboptimal efficiencies. Future riboswitch studies will benefit from comparing observed activation ratios to this simple expression for the optimal activation ratio. We present experimental and computational suggestions for how to make these quantitative comparisons and suggest new molecular mechanisms that may allow non-equilibrium riboswitches to surpass the derived limit.

Keywords: Aptamer; Biosensor; Genetic regulation; Optimality; RNA; Riboswitch; Thermodynamic model.

Fig. A1

A depiction of states and weights for a generalized nine-state model of riboswitch function.

Fig. 1

Schematics for how an RNA conformational change can induce anticooperativity or cooperativity in binding an input and output ligand, corresponding to (A) an OFF-switch and (B) an ON-switch, respectively.

Fig. 2

Minimal thermodynamic model for riboswitch function. Depicted are states and associated Boltzmann weights for (A) an OFF-switch and (B) an ON-switch.

Fig. 3

Modelling illustrates tradeoffs in riboswitch behavior. (A) As the output ligand concentration [O] is changed, there is a tradeoff between achieving high activation ratio and low K_ds to the output, i.e., high activity. (B) The relationship between the achievable $K_d^{OFF}$ and $K_d^{ON}$ is strongly dependent on the ratio of input concentration to input K_d. Within each of these curves, there is another tradeoff between achieving high activation ratio $\frac{K_d^{OFF}}{K_d^{ON}}$ and low K_ds to the output, i.e., high activity.

Fig. 4

(A) Comparison of experimental (dark bars) and theoretically optimal ARs (light bars) of natural riboswitches in literature to date, as computed using the thermodynamic model presented here, presented in order of appearance in the literature. (B) Optimalities of natural riboswitches, computed as experimental AR/theoretical AR. (C) Scatter plot of experimental and theoretical ARs. Values are only shown for cases where previous literature provide enough information to estimate both experimental and theoretically optimal ARs, see main text for caveats.

Fig. 4

(A) Comparison of experimental (dark bars) and theoretically optimal ARs (light bars) of natural riboswitches in literature to date, as computed using the thermodynamic model presented here, presented in order of appearance in the literature. (B) Optimalities of natural riboswitches, computed as experimental AR/theoretical AR. (C) Scatter plot of experimental and theoretical ARs. Values are only shown for cases where previous literature provide enough information to estimate both experimental and theoretically optimal ARs, see main text for caveats.

Fig. 5

(A) Comparison of experimental (dark bars) and theoretical ARs (light bars) of synthetic riboswitches in literature to date, presented in order of appearance in the literature. (B) Optimalities of synthetic riboswitches, computed as experimental AR/theoretical AR. (C) Scatter plot depiction of optimal vs. experimental ARs. Values are only shown for cases where previous literature provide enough information to estimate both experimental and theoretically optimal ARs, see main text for caveats.

Fig. 5

(A) Comparison of experimental (dark bars) and theoretical ARs (light bars) of synthetic riboswitches in literature to date, presented in order of appearance in the literature. (B) Optimalities of synthetic riboswitches, computed as experimental AR/theoretical AR. (C) Scatter plot depiction of optimal vs. experimental ARs. Values are only shown for cases where previous literature provide enough information to estimate both experimental and theoretically optimal ARs, see main text for caveats.