Biomolecular Receptors Research Articles

"Binding" or "Binding and Switching"? A Perspective on Resolving Conformational Changes of Surface-Attached Biomolecular Receptors.

Understanding the structural reconfiguration of a biomolecular receptor remains a topic of particular interest to the biosensing community. This is because conformationally changing receptors are commonly employed in biosensors to harness their capability to bind specifically to their target. Often, such receptors are attached to surfaces so that binding can be transduced into a measurable response. Doing so, however, can impose constraints on the possible configurations they can adopt. Such constraints can ultimately influence, for example, their receptor-target binding models or their affinity, which is essential to provide the desired analytical performances in biosensors. Motivated by the idea of gaining further insights into the impact of surface attachment on conformationally switching receptors attached to surfaces, we explore here the various surface-based techniques capable of monitoring structural changes. We decided to narrow our survey to techniques that have been applied to the investigation of nucleic acids to provide an overview of their key features. We envision that this will bring a broader perspective of the field and the challenges ahead with the hopes of "finding the switch" in surface-attached biomolecular receptors.

Stability of N-Heterocyclic Carbene Monolayers Under Continuous Voltammetric Cycling

N-heterocyclic carbenes (NHCs) are promising monolayer-forming chemistries that can overcome limitations of thiol-based monolayers in terms of stability, surface functionality and reactivity across a variety of transition metal surfaces. Recent publications have reported the ability of NHCs to support biomolecular receptors on gold substrates for sensing applications, and improved tolerance to prolonged biofluid exposure relative to thiol chemistries. However, important questions remain regarding the stability of these monolayers when subjected to voltage perturbations, which is needed for technology application in electrochemical platforms. In response, we investigated the ability of two NHC chemistries, 1,3-diisopropylbenzimidazole and 5-(ethoxycarbonyl)-1,3-diisopropylbenzimidazole, to form monolayers via self-assembly from methanolic solutions of their trifluoromethanesulfonate salts. We compared the electrochemical behavior of the resulting monolayers relative to that of benchmark mercaptohexanol monolayers in phosphate-buffered saline. In this presentation I will discuss the results of this study and how they can affect future use of NHCs in electrochemical biosensors.

Stability of N-Heterocyclic Carbene Monolayers under Continuous Voltammetric Interrogation.

N-Heterocyclic carbenes (NHCs) are promising monolayer-forming ligands that can overcome limitations of thiol-based monolayers in terms of stability, surface functionality, and reactivity across a variety of transition-metal surfaces. Recent publications have reported the ability of NHCs to support biomolecular receptors on gold substrates for sensing applications and improved tolerance to prolonged biofluid exposure relative to thiols. However, important questions remain regarding the stability of these monolayers when subjected to voltage perturbations, which is needed for applications with electrochemical platforms. Here, we investigate the ability of two NHCs, 1,3-diisopropylbenzimidazole and 5-(ethoxycarbonyl)-1,3-diisopropylbenzimidazole, to form monolayers via self-assembly from methanolic solutions of their trifluoromethanesulfonate salts. We compare the electrochemical behavior of the resulting monolayers relative to that of benchmark mercaptohexanol monolayers in phosphate-buffered saline. Within the -0.15 to 0.25 V vs Ag|AgCl voltage window, NHC monolayers are stable on gold surfaces, wherein they electrochemically perform like thiol-based monolayers and undergo similar reorganization kinetics, displaying long-term stability under incubation in buffered media and under continuous voltammetric interrogation. At negative voltages, NHC monolayers cathodically desorb from the electrode surface at lower bias (-0.1 V) than thiol-based monolayers (-0.5 V). At voltages more positive than 0.25 V, NHC monolayers anodically desorb from electrode surfaces at similar voltages to thiol-based monolayers. These results highlight new limitations to NHC monolayer stability imposed by electrochemical interrogation of the underlying gold electrodes. Our results serve as a framework for future optimization of NHC monolayers on gold for electrochemical applications, as well as structure-functionality studies of NHCs on gold.

UV-Resonance Raman Spectra of Systems in Complex Environments: A Multiscale Modeling Applied to Doxorubicin Intercalated into DNA.

UV-Resonance Raman (RR) spectroscopy is a valuable tool to study the binding of drugs to biomolecular receptors. The extraction of information at the molecular level from experimental RR spectra is made much easier and more complete thanks to the use of computational approaches, specifically tuned to deal with the complexity of the supramolecular system. In this paper, we propose a protocol to simulate RR spectra of complex systems at different levels of sophistication, by exploiting a quantum mechanics/molecular mechanics (QM/MM) approach. The approach is challenged to investigate RR spectra of a widely used chemotherapy drug, doxorubicin (DOX) intercalated into a DNA double strand. The computed results show good agreement with experimental data, thus confirming the reliability of the computational protocol.

On the Rational Design of Cooperative Receptors.

Cooperativity (homotropic allostery) is the primary mechanism by which evolution steepens the binding curves of biomolecular receptors to produce more responsive input-output behavior in biomolecular systems. Motivated by the ubiquity with which nature employs this effect, over the past 15 years we, together with other groups, have engineered this mechanism into several otherwise noncooperative receptors. These efforts largely aimed to improve the utility of such receptors in artificial biotechnologies, such as synthetic biology and biosensors, but they have also provided the first quantitative, experimental tests of longstanding ideas about the mechanisms underlying cooperativity. In this article, we review the literature on the design of this effect, paying particular attention to the design strategies involved, the extent to which each can be rationally applied to (and optimized for) new receptors, and what each teaches us about the origins and optimization of this important phenomenon.

Protein Flexibility and Dissociation Pathway Differentiation Can Explain Onset of Resistance Mutations in Kinases**

AbstractUnderstanding how mutations render a drug ineffective is a problem of immense relevance. Often the mechanism through which mutations cause drug resistance can be explained purely through thermodynamics. However, the more perplexing situation is when two proteins have the same drug binding affinities but different residence times. In this work, we demonstrate how all‐atom molecular dynamics simulations using recent developments grounded in statistical mechanics can provide a detailed mechanistic rationale for such variances. We discover dissociation mechanisms for the anti‐cancer drug Imatinib (Gleevec) against wild‐type and the N368S mutant of Abl kinase. We show how this point mutation triggers far‐reaching changes in the protein's flexibility and leads to a different, much faster, drug dissociation pathway. We believe that this work marks an efficient and scalable approach to obtain mechanistic insight into resistance mutations in biomolecular receptors that are hard to explain using a structural perspective.

Protein Flexibility and Dissociation Pathway Differentiation Can Explain Onset of Resistance Mutations in Kinases.

Understanding how mutations render a drug ineffective is a problem of immense relevance. Often the mechanism through which mutations cause drug resistance can be explained purely through thermodynamics. However, the more perplexing situation is when two proteins have the same drug binding affinities but different residence times. In this work, we demonstrate how all-atom molecular dynamics simulations using recent developments grounded in statistical mechanics can provide a detailed mechanistic rationale for such variances. We discover dissociation mechanisms for the anti-cancer drug Imatinib (Gleevec) against wild-type and the N368S mutant of Abl kinase. We show how this point mutation triggers far-reaching changes in the protein's flexibility and leads to a different, much faster, drug dissociation pathway. We believe that this work marks an efficient and scalable approach to obtain mechanistic insight into resistance mutations in biomolecular receptors that are hard to explain using a structural perspective.

(Invited) Diamond Based Chemical/Biochemical Sensors: State-of-the-Art and Perspectives

Diamond is grown in some laboratories by either HPHT process or Plasma-Enhanced Chemical Vapor Deposition (MP-CVD) since a few decades. Single crystal diamond exhibits outstanding properties including a high optical transparency over a broad electromagnetic spectrum, high thermal conductivity approx. five times higher than copper, and acoustic wave velocity close to 19 000 m.s-1. It displays also remarkable mechanical properties with e.g. a Young’s modulus exceeding 1000 GPa along with high resistance to fracture, to name a few. Some of these properties remain also remarkable in its polycrystalline form when compare to most other materials. Furthermore, diamond can be doped with nitrogen or boron during growth, offering electrical properties from semiconducting to quasi-metallic regimes. When heavily doped with boron (~2.1021 cm-3), the so-called Boron Doped Diamond (BDD) electrodes become attractive electrodes featuring a high potential window > 3V in water and low double-layer capacitance. Moreover, diamond is extremely resilient to corrosion and more generally to chemical attacks. It is also biocompatible, which makes it very attractive for in-vivo sensing applications. Finally, the carbon nature of the diamond offers wide opportunities for surface grafting of chemical or biochemical functional groups through highly stable covalent carbon-carbon bonding. These properties can be exploited advantageously to enhance the analytical performances and stability of chemical/biochemical sensors and have motivated our research over the last 15 years. Our work focuses mainly on polycrystalline diamond thin films that can be grown typically on inches silicon substrates, thus offering access to some clean-room processes and potentially large-scale production.Several processes were elaborated to micro-pattern diamond layers in order to design chemical transducers such as gravimetric MEMS devices, electrodes, field effect transistors, etc. Diamond microstructures may also be transferred to flexible parylene or polyimide substrates, thus making them attractive e.g. for wearable sensors and implantable medical electrodes. Further techniques have also been developed to enhance the active surface area of diamond transducer surfaces at the nanoscale and thus increase drastically the sensitivity of the resulting sensors by multiplying the number of active sites. Here diamond is typically grown onto well-chosen high aspect ratio templates that can withstand the growth conditions of diamond in high-density hydrogen plasma. Most of these methods have been “standardized”. They involve clean-room processing including dry etching, photolithography, and so on and forth. As examples, heavily doped diamond electrodes were developed successfully both as macro- and micro-electrodes for biomedical, pharmaceutical or foodstuff analysis applications. These applications benefit both from the high analytical performances of diamond electrodes in particular due to their low background signals and high reactivity, and high stability and reliability. BDD electrodes may also be modified with transition metal nanoparticles to enhance their catalytic behavior. From this concept, diamond multi-electrode arrays were designed for chemical patterns identification, for instance for sensory analysis of coffee, or for the monitoring of environmental pollutants. BDD electrodes offer also significant advantages in electrochemiluminescence (ECL) techniques, which are being investigated for various applications ranging from foodstuff analysis to narcotics detection. A key benefit of BDD electrodes for all of the above applications is certainly that they can be electrochemically reactivated following fouling, sometimes directly in the analytical medium, to maintain high reactivity thus opening the way to reusable sensors and online monitoring. Besides, BDD microelectrode arrays have also been transferred to flexible substrates for neural stimulation and recording, along with in-vivo neuromodulators measurements. Finally, diamond based MEMS devices (microcantilevers, SAW sensors) take advantage both of the mechanical properties of diamond, along with steady carbon interface for convenient bio-functionalization. Our work here focused mainly on the detection of odorant molecules, using biomolecular receptors involved in olfaction in Nature as sensitive layers, including Odorant Binding Proteins (OBPs), Major Urinary Proteins (MUPs) and Olfactory Receptors (OR). Multisensor array instrumentations were developed around this concept, for applications ranging from breathe analysis to security applications.

Rational design to control the trade-off between receptor affinity and cooperativity

Cooperativity enhances the responsiveness of biomolecular receptors to small changes in the concentration of their target ligand, albeit with a concomitant reduction in affinity. The binding midpoint of a two-site receptor with a Hill coefficient of 1.9, for example, must be at least 19 times higher than the dissociation constant of the higher affinity of its two binding sites. This trade-off can be overcome, however, by the extra binding energy provided by the addition of more binding sites, which can be used to achieve highly cooperative receptors that still retain high affinity. Exploring this experimentally, we have employed an "intrinsic disorder" mechanism to design two cooperative, three-binding-site receptors starting from a single-site-and thus noncooperative-doxorubicin-binding aptamer. The first receptor follows a binding energy landscape that partitions the energy provided by the additional binding event to favor affinity, achieving a Hill coefficient of 1.9 but affinity within a factor of 2 of the parent aptamer. The binding energy landscape of the second receptor, in contrast, partitions more of this energy toward cooperativity, achieving a Hill coefficient of 2.3, but at the cost of 4-fold poorer affinity than that of the parent aptamer. The switch between these two behaviors is driven primarily by the affinity of the receptors' second binding event, which serves as an allosteric "gatekeeper" defining the extent to which the system is weighted toward higher cooperativity or higher affinity.

(Invited) Smart Material for Smart Sensors: Addressing Future Challenges in Bio/Chemical Sensing with Diamond

Introduction Synthetic diamond can be grown in the laboratory by Plasma-Enhanced Chemical Vapor Deposition (MP-CVD) both as single crystals or polycrystalline thin films [1]. This material exhibits outstanding physical and chemical properties, including typically a high optical transparency over a broad electromagnetic spectrum, high thermal conductivity (~five times higher than copper) and acoustic wave velocity close to 19 000 m.s-1. It displays also remarkable mechanical properties with e.g. a Young’s modulus exceeding 1000 GPa and high resistance to fracture. Diamond may also be doped with boron during growth, offering electrical properties from semiconducting to quasi-metallic regimes. When heavily doped with boron, it becomes an exceptional electrode material featuring a high potential window > 3V in aqueous environments, and low capacitive background current. Moreover, diamond is extremely resilient to chemical attacks, and corrosion, and several studies have shown that it is a biocompatible material, which makes it very attractive for in-vivo sensing applications. Finally, the carbon terminated surface of diamond layers enables grafting of a wide range of chemical or biochemical functional groups through highly stable C-C covalent bonding. All those properties, which can be advantageously exploited to enhance the analytical performances and stability of chemical/biochemical sensors, have motivated our research over the last decade. Diamond-based sensor fabrication methods Polycrystalline diamond thin films are generally grown by MP-CVD in a hydrogen plasma, and in the presence of methane as the source of carbon. Several processes have been developed to micro-pattern diamond layers, either by top-up or top-down approaches, in order to design chemical transducers technologies including gravimetric MEMS devices [2], micro-electrodes or IDT electrodes [3], Field Effect Transistors, etc. (Fig.1). Some of these microstructures may also be transferred to flexible substrates like parylene or polyimide [4], thus making them attractive e.g. for wearable sensors. Further techniques have also been developed to enhance the active surface area of diamond transducer surfaces and thus the sensitivity of the resulting sensors. To this end, diamond can be grown for instance onto high aspect ratio templates such as vertically aligned CNTs or on some well selected high-porosity polymers. Most of these methods have been “standardized” and involve clean-room processing including dry etching, photolithography, and so forth. Results and applications Bare diamond electrodes, when heavily doped with boron (~2.1021 cm-3), have been developed successfully both as macro- and micro-electrodes for biomedical (e.g. detection of uric acid in human urine) or pharmaceutical applications. These applications benefit here both from the high analytical performances of diamond electrodes, and high stability and reliability. In particular they can be electrochemically reactivated following fouling, sometimes directly in the analytical medium, to maintain high reactivity thus opening the way to reusable sensors and online monitoring. Such electrodes may also be functionalized by transition metal nanoparticles to enhance the catalytic behavior of the electrodes. From this concept, diamond multi-electrode arrays may be designed for chemical patterns identification. This approach was used for instance for coffee capsule discrimination [5]. Several assets of boron doped diamond electrodes were also be used in combination with electrochemiluminescence (ECL) techniques for foodstuff analysis, namely the selective detection of skatole in pig fat in the context of boar taint detection. Diamond microelectrode arrays have also been developed on flexible substrates for neural stimulation and recording, along with in-vivo neuromodulators measurements.Besides, a range of diamond based MEMS devices were developed (microcantilevers, SAW sensors), taking advantage both of the mechanical properties of diamond, along with steady carbon interface for convenient bio-functionalization. Our studies focused mainly on odor detection, using biomolecular receptors involved in olfaction in Nature as sensitive layers, including Odorant Binding Proteins (OBPs), Major Urinary Proteins (MUPs) and Olfactory Receptors (OR). Multisensor array instrumentations were developed around this concept, for applications ranging from security applications or breathe analysis.

Optimizing the Specificity Window of Biomolecular Receptors Using Structure-Switching and Allostery.

To ensure maximum specificity (i.e., minimize cross-reactivity with structurally similar analogues of the desired target), most bioassays invoke "stringency", the careful tuning of the conditions employed (e.g., pH, ionic strength, or temperature). Willingness to control assay conditions will fall, however, as quantitative, single-step biosensors begin to replace multistep analytical processes. This is especially true for sensors deployed in vivo, where the tuning of such parameters is not just inconvenient but impossible. In response, we describe here the rational adaptation of two strategies employed by nature to tune the affinity of biomolecular receptors so as to optimize the placement of their specificity "windows" without the need to alter measurement conditions: structure-switching and allosteric control. We quantitatively validate these approaches using two distinct, DNA-based receptors: a simple, linear-chain DNA suitable for detecting a complementary DNA strand and a structurally complex DNA aptamer used for the detection of a small-molecule drug. Using these models, we show that, without altering assay conditions, structure-switching and allostery can tune the concentration range over which a receptor achieves optimal specificity over orders of magnitude, thus optimally matching the specificity window with the range of target concentrations expected to be seen in a given application.

Multisegment nanowire/nanoparticle hybrid arrays as electrochemical biosensors for simultaneous detection of antibiotics

Antibiotics such as penicillin and tetracycline drugs are widely used in food animals to treat, control, and prevent diseases, and penicillin is approved for use to improve growth rates in pigs and poultry. However, due to the overuse of antibiotics in food and medical industry, the antimicrobial resistance is starting to show up in some developing countries. The antibiotic abuse may cause allergic reactions, resistance in microorganisms and general lowering of immunity in consumers of meat and dairy products. It is important and necessary to develop an easy, inexpensive, and quantitative sensing method to monitor and analyze the antibiotics concentration in real samples such as milk or meat. In this research, an electrochemical biosensor based on hybrid nanowire/nanoparticle array with various bio-molecular receptors was fabricated for the simultaneous detection of penicillin and tetracycline. The vertically aligned Pt-Au nanowire array has been prepared by an electrodeposition method within anodic aluminum oxide (AAO) membranes; L-cysteine was used to form a monolayer on the Au segment as the bio-receptor for tetracycline detection; electroless plating of Au nanoparticles was applied on the Pt nanowire segments, and then the penicillinase was immobilized on the Au nanoparticles using EDC/NHS cross-linker. The prepared Au(L-cysteine)-Pt(penicillinase) nanowire array electrode showed simultaneous detection ability and remarkably high sensitivity of penicillin and tetracycline, which are 41.2 μA μM−1 cm−2 for penicillin detection and 26.4 μA μM−1 cm−2 for tetracycline detection. The sensitivities of each analytes with different segment length were also investigated. Real sample tests with chicken and beef extract were conducted, which showed good recovery performance. Due to the advantages of the hybrid nanowire/nanoparticle array structure, this new sensor can serve as an enhanced platform for simultaneous detection of various bioanalytes.

Tunable DNA Hybridization Enables Spatially and Temporally Controlled Surface-Anchoring of Biomolecular Cargo.

The controlled immobilization of biomolecules onto surfaces is relevant in biosensing and cell biological research. Spatial control is achieved by surface-tethering molecules in micro- or nanoscale patterns. Yet, there is an increasing demand for temporal control over how long biomolecular cargo stays immobilized until released into the medium. Here, we present a DNA hybridization-based approach to reversibly anchor biomolecular cargo onto micropatterned surfaces. Cargo is linked to a DNA oligonucleotide that hybridizes to a sequence-complementary, surface-tethered strand. The cargo is released from the substrate by the addition of an oligonucleotide that disrupts the duplex interaction via toehold-mediated strand displacement. The unbound tether strand can be reloaded. The generic strategy is implemented with small-molecule or protein cargo, varying DNA sequences, and multiple surface patterning routes. The approach may be used as a tool in biological research to switch membrane proteins from a locally fixed to a free state, or in biosensing to shed biomolecular receptors to regenerate the sensor surface.

Triple-Helix Molecular Switch Electrochemical Ratiometric Biosensor for Ultrasensitive Detection of Nucleic Acids

Biomolecular receptors such as nucleic acids that switch between two or more conformations upon binding to a specific target can be used to build specific and sensitive biosensors. In this work, based on the electrochemical dual-signaling ratiometric strategy and triple-helix molecular switch, we developed a selective, reusable, and simple electrochemical DNA (E-DNA) biosensor for target DNA (T-DNA) detection. A hairpin DNA capture probe labeled with methylene blue (MB-DNA) self-assembles on the surface of a gold electrode (GE) through Au-S bond, and then a single-strand DNA modified with two ferrocenes (Fc-DNA) on each end to enhance the oxidation signal hybridizes with the MB-DNA to form a triple-helix conformation. When T-DNA exists, the Fc-DNA hybridizes with T-DNA disassembling the triple-helix stem and allowing the MB-DNA to revert to its hairpin structure. Hence, the Fc tags diffuse away from the GE surface while the MB tags remain affixed close to it, resulting in a decrease in the peak current of Fc (IFc) and an increase in that of MB (IMB). The linear relationship between the value of IMB/IFc and the T-DNA concentration is observed from 0.5 to 80 pM, and the limit of detection is as low as 0.12 pM. The developed E-DNA biosensor may have great potential in the electrochemical detection of a wide range of analytes and be a biosensing platform for early clinical diagnosis and biomedical research.

Stereochemical plasticity modulates cooperative binding in a CoII12L6 cuboctahedron.

Biomolecular receptors are able to process information by responding differentially to combinations of chemical signals. Synthetic receptors that are likewise capable of multi-stimuli response can form the basis of programmable molecular systems, wherein specific input sequences create distinct outputs. Here we report a pseudo-cuboctahedral assembly capable of cooperatively binding anionic and neutral guest species. The binding of pairs of fullerene guests was observed to effect the all-or-nothing cooperative templation of an S6-symmetric host stereoisomer. This bis-fullerene adduct exhibits different cooperativity in binding pairs of anions from the fullerene-free parent: in one case, positive cooperativity is observed, while in another all binding affinities are enhanced by an order of magnitude, and in a third the binding events are only minimally perturbed. This intricate modulation of binding affinity, and thus cooperativity, renders our new cuboctahedral receptor attractive for incorporation into systems with complex, programmable responses to different sets of stimuli.

Using Nature's "Tricks" To Rationally Tune the Binding Properties of Biomolecular Receptors.

The biosensor community has long focused on achieving the lowest possible detection limits, with specificity (the ability to differentiate between closely similar target molecules) and sensitivity (the ability to differentiate between closely similar target concentrations) largely being relegated to secondary considerations and solved by the inclusion of cumbersome washing and dilution steps or via careful control experimental conditions. Nature, in contrast, cannot afford the luxury of washing and dilution steps, nor can she arbitrarily change the conditions (temperature, pH, ionic strength) under which binding occurs in the homeostatically maintained environment within the cell. This forces evolution to focus at least as much effort on achieving optimal sensitivity and specificity as on achieving low detection limits, leading to the "invention" of a number of mechanisms, such as allostery and cooperativity, by which the useful dynamic range of receptors can be tuned, extended, narrowed, or otherwise optimized by design, rather than by sample manipulation. As the use of biomolecular receptors in artificial technologies matures (i.e., moves away from multistep, laboratory-bound processes and toward, for example, systems supporting continuous in vivo measurement) and these technologies begin to mimic the reagentless single-step convenience of naturally occurring chemoperception systems, the ability to artificially design receptors of enhanced sensitivity and specificity will likely also grow in importance. Thus motivated, we have begun to explore the adaptation of nature's solutions to these problems to the biomolecular receptors often employed in artificial biotechnologies. Using the population-shift mechanism, for example, we have generated nested sets of receptors and allosteric inhibitors that greatly expanded the normally limited (less than 100-fold) useful dynamic range of unmodified molecular and aptamer beacons, enabling the single-step (e.g., dilution-free) measurement of target concentrations across up to 6 orders of magnitude. Using this same approach to rationally introduce sequestration or cooperativity into these receptors, we have likewise narrowed their dynamic range to as little as 1.5-fold, vastly improving the sensitivity with which they respond to small changes in the concentration of their target ligands. Given the ease with which we have been able to introduce these mechanisms into a wide range of DNA-based receptors and the rapidity with which the field of biomolecular design is maturing, we are optimistic that the use of these and similar naturally occurring regulatory mechanisms will provide viable solutions to a range of increasingly important analytical problems.

Fluorine nuclear magnetic resonance-based assay in living mammalian cells

Nuclear magnetic resonance (NMR)-based screening has been recognized as a powerful approach for the identification and characterization of molecules interacting with pharmaceutical targets. Indeed, several NMR methods have been developed and successfully applied to many drug discovery projects. Whereas most of these approaches have targeted isolated biomolecular receptors, very few cases are reported with the screening performed in intact cells and cell extracts. Here we report the first successful application of the fluorine NMR-based assay n-FABS (n-fluorine atoms for biochemical screening) in living mammalian cells expressing the membrane protein fatty acid amide hydrolase (FAAH). This method allows the identification of both weak and potent inhibitors and the measurement of their potency in a physiological environment.

A Disorder-Based Strategy for the Introduction of Allosteric, “Hill-Type” Cooperativity into Artificial Receptors

Control over of the sensitivity with which biomolecular receptors respond to small changes in target molecule concentration is crucial to many biological processes. The ability to introduce cooperativity into artificial bioreceptors would likely prove useful for applications such as biosensors, genetic logic gates, and “smart” materials, in which highly responsive behavior is of value. In Nature, this control is commonly achieved via allosteric, “Hill-type,” cooperativity, in which binding events on a multivalent receptor are coupled so that the first event enhances the affinity of subsequent events, producing an “all-or-nothing” binding response and, in turn, a higher-order, steeper, dependence on target molecule concentration. Here we use an intrinsic-disorder-mediated approach to rationally and quantitatively introduce this useful property into several normally non-cooperative biomolecular receptors. Specifically, we fabricate a tandem repeat of the receptor that is destabilized by the inclusion of a disordered loop. The first target binding event pays an entropic cost to close this loop, thus forming a structured site for a second target molecule to bind with comparatively higher affinity. By changing the length of the loop, we can quantitatively change the energetic cost of closing the loop and in turn the extent of cooperativity, and thus the order of the binding curve and sensitivity to small changes in concentration. Using this approach we have rationally introduced cooperativity into three unrelated aptamers, achieving in the best of these a Hill coefficient experimentally indistinguishable from the theoretically expected maximum. Furthermore, 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.

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

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 "Hill-type" 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 intrinsic-disorder-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.

Using the population-shift mechanism to rationally introduce "Hill-type" cooperativity into a normally non-cooperative receptor.

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 low-affinity 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.