Abstract
Our ability to recreate complex biochemical mechanisms in designed, artificial systems provides a stringent test of our understanding of these mechanisms and opens the door to their exploitation in artificial biotechnologies. Motivated by this philosophy, here we have recapitulated in vitro the “target sequestration” mechanism used by nature to improve the sensitivity (the steepness of the input/output curve) of many regulatory cascades. Specifically, we have employed molecular beacons, a commonly employed optical DNA sensor, to recreate the sequestration mechanism and performed an exhaustive, quantitative study of its key determinants (e.g., the relative concentrations and affinities of probe and depletant). We show that, using sequestration, we can narrow the pseudo-linear range of a traditional molecular beacon from 81-fold (i.e., the transition from 10% to 90% target occupancy spans an 81-fold change in target concentration) to just 1.5-fold. This narrowing of the dynamic range improves the sensitivity of molecular beacons to that equivalent of an oligomeric, allosteric receptor with a Hill coefficient greater than 9. Following this we have adapted the sequestration mechanism to steepen the binding-site occupancy curve of a common transcription factor by an order of magnitude over the sensitivity observed in the absence of sequestration. Given the success with which the sequestration mechanism has been employed by nature, we believe that this strategy could dramatically improve the performance of synthetic biological systems and artificial biosensors.
Highlights
In order to test the extent to which we understand complex biochemical systems -and to improve our ability to exploit them in man-made technologies- it is important to reconstruct these processes in the laboratory
Low concentrations of a given target molecule are sequestered by binding to a high affinity receptor that acts as a ‘‘depletant,’’ which serves as a ‘‘sink’’ that prevents the accumulation of free target without generating an output signal (Figure 1a)
Synthetic biomolecular switches developed by Kramer and coworkers for the detection of specific DNA or RNA sequences [29], are widely used in the diagnosis of genetic and infectious diseases [30,31,32]
Summary
In order to test the extent to which we understand complex biochemical systems -and to improve our ability to exploit them in man-made technologies (e.g., synthetic biology; biosensors)- it is important to reconstruct these processes in the laboratory Illustrative examples of this include recent demonstrations of synthetic genetic networks in which genetic elements are ‘‘mixed and matched’’ in order to create artificial bistable ‘‘toggle switches,’’ genetic oscillators and other complex, non-linear input/output behaviors (e.g., [1,2,3,4,5]). The rapidly rising concentration of free target binds to –and activates– a second, lower affinity (higher dissociation constant) receptor (or ‘‘probe’’) that, unlike the depletant, generates an output signal This threshold effect generates a ‘‘pseudo-cooperative’’ dose-response curve, which is much more sensitive (much steeper) than the hyperbolic ‘‘Langmuir isotherm’’ produced by simple, single site binding (Figure 1b, Bottom) [17,18,19]. The sensitivity of biological systems, such as metabolic networks or signal transduction pathways, is defined as the ratio of
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