Intrinsic disorder as a generalizable strategy for the rational design of highly responsive, allosterically cooperative receptors

Abstract

Control over the sensitivity with which biomolecular receptors respond to small changes in the concentration of their target ligand is critical for the proper function of many cellular processes. Such control could likewise be of utility in artificial biotechnologies, such as biosensors, genetic logic gates, and "smart" materials, in which highly responsive behavior is of value. In nature, the control of molecular responsiveness is often achieved using "Hilltype" cooperativity, a mechanism in which sequential binding events on a multivalent receptor are coupled such that the first enhances the affinity of the next, producing a steep, higher-order dependence on target concentration. Here, we use an intrinsicdisorder-based mechanism that can be implemented without requiring detailed structural knowledge to rationally introduce this potentially useful property into several normally noncooperative biomolecules. To do so, we fabricate a tandem repeat of the receptor that is destabilized (unfolded) via the introduction of a long, unstructured loop. The first binding event requires the energetically unfavorable closing of this loop, reducing its affinity relative to that of the second binding event, which, in contrast occurs at a preformed site. Using this approach, we have rationally introduced cooperativity into three unrelated DNA aptamers, achieving in the best of these a Hill coefficient experimentally indistinguishable from the theoretically expected maximum. The extent of cooperativity and thus the steepness of the binding transition are, moreover, well modeled as simple functions of the energetic cost of binding-induced folding, speaking to the quantitative nature of this design strategy. ultrasensitivity | intrinsically disordered proteins | biosensors | synthetic biology | ribozymes

Real-time measurement of small molecules directly in awake, ambulatory animals

“…Created using well-established in vitro selection methods (16,17), aptamers can be generated that bind a wide range of analytes (18) and can be rationally reengineered such that they undergo a large-scale conformational change upon binding these analytes (19) over arbitrarily broad (20,21) or narrow (20,22) concentration windows. E-AB sensors use this conformational change to generate an easily measureable electrochemical signal without the need for the target to undergo a chemical transformation (23).…”

Using Nature’s “Tricks” To Rationally Tune the Binding Properties of Biomolecular Receptors

“…We demonstrate this approach using an otherwise noncooperative (bottom, left) cocaine-binding aptamer and achieved a cooperative response covering just 13-fold (bottom right). Figure adapted with permission from ref 53. Copyright 2014 National Academy of Sciences.…”

Sensitive Detection of Small-Molecule Targets Using Cooperative Binding Split Aptamers and Enzyme-Assisted Target Recycling

“…This implies that a transition of CBSA assembly from 10% to 90% requires only a 16-fold increase in target concentration, compared to its parent split aptamers which require an 81-fold increase. 28 NUPACK analysis 37 of the DIS-binding CBSA sequences demonstrated that both fragments are predominantly un-assembled (99%) in the absence of target under our experimental conditions, but DIS-SF exists primarily as a duplexed structure (Supporting Information, Figure S3A). Therefore, binding of the first target to DIS–CBSA-4536 should open up the DIS-SF duplex, facilitating binding of a second DIS molecule to the CBSA, This results in a high level of cooperativity.…”