Using the Population‐Shift Mechanism to Rationally Introduce “Hill‐type” Cooperativity into a Normally Non‐Cooperative Receptor

Abstract

Allosteric cooperativity, which nature uses to improve the sensitivity with which biomolecular receptors respond to small changes in ligand concentration, could likewise be of use in improving the responsiveness of artificial biosystems. Thus motivated, we demonstrate here the rational design of cooperative molecular beacons, a widely employed DNA sensor, using a generalizable population-shift approach in which we engineer receptors that equilibrate between a lowaffinity state and a high-affinity state exposing two binding sites. Doing so we achieve cooperativity within error of ideal behavior, greatly steepening the beacon s binding curve relative to that of the parent receptor. The ability to rationally engineer cooperativity should prove useful in applications such as biosensors, synthetic biology and "smart" biomaterials, in which improved responsiveness is of value.The ability to respond sensitively to small changes in a molecular input is critical to many biological processes. This ability allows cells and organisms to react to subtle molecular cues and to convert complex input signals into decisive, effectively binary outputs. [1] An enhanced ability to detect small changes in molecular concentration would likely also prove of value in many biotechnologies. The ratio between an effective dose and a toxic dose of some drugs, for example, can be as little as 4-fold, [2] and thus to measure these with clinically relevant precision requires sensors that respond robustly to small changes in drug concentration.Driven by the advantages associated with enhanced molecular responsiveness evolution has invented a number of mechanisms, including sequestration, amplification cascades, and receptor co-localization, by which the relative insensitivity of single-site receptors (e.g., they require an 81-fold concentration change to transition from 10 % to 90 % occupancy) can be overcome. [1] To date many of these mechanisms have been exploited to improve the responsiveness of biotechnologies ranging from molecular [3] and genetic [4] logic gates to ultra-responsive biosensors [5,6] and digital, "all-or-none" drug-delivery systems. [7,8] Allosteric cooperativity, however, which is arguably the simplest solution to this problem, [9,10] has seen adaptation to only a handful of small-molecule [11,12] and biopolymer-based receptors. [13][14][15][16] Here we explore and articulate design principles underlying this mechanism by engineering it into a normally noncooperative receptor, thus improving the receptor s ability to respond to subtle concentration changes.The occupancy of an allosterically cooperative receptor goes aswhere K Half is the concentration at which half of all binding sites are occupied and n H , the "Hill coefficient," provides a convenient metric of cooperativity: a system is noncooperative at n H = 1, and approaches maximum cooperativity as n H approaches the number of binding sites on the receptor. [17] (Note: here we discuss positive cooperativity, which steepens the binding curve. Negative coope...

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

“…Unfortunately, however, although nature frequently uses the simple, elegant mechanism of allosteric cooperativity to overcome this limitation, the generalizable ability to recapitulate this behavior in normally noncooperative biomolecular receptors has hitherto remained elusive, with successful examples of artificially engineered, allosteric cooperativity having been restricted to a small number of more-or-less nongeneralizable examples (9)(10)(11)(12). In part, this is because our ability to rationally design biomolecules that switch reversibly between two welldefined conformations likewise remains limited (22).…”

“…The design of allosterically cooperative receptors, in contrast, has seen far less success. That is, although a handful of examples of rationally designed cooperativity have been reported to date (9)(10)(11)(12), no general approach has previously been reported by which such behavior can be rationally introduced into any arbitrarily complex biomolecule. This failure has limited the extent to which cooperativity, which could provide a powerful means of improving the ability of artificial biotechnologies to respond to Significance Control over the sensitivity with which biomolecular receptors respond to small changes in the concentration of their target ligand is crucial to many cellular processes and likely could be of value in many biotechnologies.…”

“…This is again in contrast to heterotropic allostery, in which each binding site is chemically distinct, allowing each to be independently optimized. Given these difficulties, and given the relative infancy of biomolecular design efforts (20)(21)(22), the ability to perform the structure-based design of cooperativity appears beyond current capabilities except for the simplest, most well-understood receptors (9)(10)(11)(12). Here, however, we use an approach to the rational engineering of allosterically cooperative receptors that does not require detailed, structure-based design.…”

First Characterization of a Biphasic, Switch-like DNA Amplification

“…After exiting the first plateau many of the templates exhibit rapid second phase kinetics, marked by a large jump in reaction product that can exceed first phase reaction kinetics. It is possible that second phase could be driven by homotropic allosteric cooperativity 17 ; the trigger can bind either toehold as seen in Figure 2. The template loop structure is stable when compared to the trigger:template association (Table SI 2), and the accumulation of reaction products may possibly shift templates to an open, amplification competent state and produce nonlinear reaction kinetics.…”

“…Structure-switching sensors such as aptamers 13 and molecular beacons 14,15 change conformation in the presence of a specific target molecule. When properly designed, structure-switching biosensors can also create Hill-type ultrasensitive kinetics: biosensors with two cooperative binding sites produce an ultrasensitive response if the affinity of the target for the second site is altered by target association to the first site [16][17][18][19] . These exciting biomimetic systems typically produce outputs with nanomolar trigger inputs.…”