High-Precision, In Vitro Validation of the Sequestration Mechanism for Generating Ultrasensitive Dose-Response Curves in Regulatory Networks
Figure 2
Ultrasensitivity is a strong function of the ratio [dep]/Kdprobe.
Using molecular beacons, we have recapitulated the sequestration mechanism in vitro and, in so doing, have vastly increased the sensitivity of this commonly employed biosensor. (Left) We find that ultrasensitivity is a strong function of the ratio [dep]/Kdprobe, which measures the extent to which the concentration of the depletant rises above the affinity of the probe. (Here we are using a depletant/probe pair for which Kdprobe/Kddep = 60). To quantify the sensitivity of these dose response curves we have fitted them to the Hill equation (dotted lines) to define pseudo-Hill coefficients. (Right) This pseudo-Hill coefficient increases monotonically as the [dep]/Kdprobe ratio increases, reaching 9.4 at the highest ratios we have investigated. It is important to note, however, that although the Hill coefficient provides an easy way to compare sensitivity across different systems, the Hill equation is not a correct physical description of our system. Instead, the behavior of our system is described by the sequestration model as expressed in equation 2 (see text; see also Buchler et al., 2009). Using equation 2 and the previously determined dissociation constants of our probe and depletant [33], we can model the sensitivity of this system quantitatively (solid lines, left panel) without the use of any floating parameters. The theoretically modeled pseudo-Hill coefficient likewise describes our data quite well (solid line, right panel), deviating only slightly at our highest ratios we have investigated.