Bimodal brush-functionalized nanoparticles selective to receptor surface density.

Nanoparticles or drug carriers which can selectively bind to cells expressing receptors above a certain threshold surface density are very promising for targeting cells overexpressing specific receptors under pathological conditions. Simulations and theoretical studies have suggested that such selectivity can be enhanced by functionalizing nanoparticles with a bimodal polymer monolayer (BM) containing shorter ligated chains and longer inert protective chains. However, a systematic study of the effect of these parameters under tightly controlled conditions is still missing. Here, we develop well-defined and highly specific platforms mimicking particle-cell interface using surface chemistry to provide a experimental proof of such selectivity. Using surface plasmon resonance and atomic force microscopy, we report the selective adsorption of BM-functionalized nanoparticles, and especially, a significant enhanced selective behavior by using a BM with longer protective chains. Furthermore, a model is also developed to describe the repulsive contribution of the protective brush to nanoparticle adsorption. This model is combined with super-selectivity theory to support experimental findings and shows that the observed selectivity is due to the steric energy barrier which requires a high number of ligand-receptor bonds to allow nanoparticle adsorption. Finally, the results show how the relative length and molar ratio of two chains can be tuned to target a threshold surface density of receptors and thus lay the foundation for the rational design of BM-functionalized nanoparticles for selective targeting.

Monitoring protein conformational changes using fluorescent nanoantennas.

Understanding the relationship between protein structural dynamics and function is crucial for both basic research and biotechnology. However, methods for studying the fast dynamics of structural changes are limited. Here, we introduce fluorescent nanoantennas as a spectroscopic technique to sense and report protein conformational changes through noncovalent dye-protein interactions. Using experiments and molecular simulations, we detect and characterize five distinct conformational states of intestinal alkaline phosphatase, including the transient enzyme-substrate complex. We also explored the universality of the nanoantenna strategy with another model protein, Protein G and its interaction with antibodies, and demonstrated a rapid screening strategy to identify efficient nanoantennas. These versatile nanoantennas can be used with diverse dyes to monitor small and large conformational changes, suggesting that they could be used to characterize diverse protein movements or in high-throughput screening applications.

Design and Thermodynamics Principles to Program the Cooperativity of Molecular Assemblies.

Most functional nanosystems in living organisms are constructed using multimeric assemblies that provide multiple advantages over their monomeric counterparts such as cooperative or anti-cooperative responses, integration of multiple signals and self-regulation. Inspired by these natural nanosystems, chemists have been synthesizing self-assembled supramolecular systems over the last 50 years with increasing complexity with applications ranging from biosensing, drug delivery, synthetic biology, and system chemistry. Although many advances have been made concerning the design principles of novel molecular architectures and chemistries, little is still known, however, about how to program their dynamic of assembly so that they can assemble at the required concentration and with the right sensitivity. Here, we used synthetic DNA assemblies and double-mutant cycle analysis to explore the thermodynamic basis to program the cooperativity of molecular assemblies. The results presented here exemplify how programmable molecular assemblies can be efficiently built by fusing interacting domains and optimizing their compaction. They may also provide the rational basis for understanding the thermodynamic and mechanistic principles driving the evolution of multimeric biological complexes.

Programing Chemical Communication: Allostery vs Multivalent Mechanism.

The emergence of life has relied on chemical communication and the ability to integrate multiple chemical inputs into a specific output. Two mechanisms are typically employed by nature to do so: allostery and multivalent activation. Although a better understanding of allostery has recently provided a variety of strategies to optimize the binding affinity, sensitivity, and specificity of molecular switches, mechanisms relying on multivalent activation remain poorly understood. As a proof of concept to compare the thermodynamic basis and design principles of both mechanisms, we have engineered a highly programmable DNA-based switch that can be triggered by either a multivalent or an allosteric DNA activator. By precisely designing the binding interface of the multivalent activator, we show that the affinity, dynamic range, and activated half-life of the molecular switch can be programed with even more versatility than when using an allosteric activator. The simplicity by which the activation properties of molecular switches can be rationally tuned using multivalent assembly suggests that it may find many applications in biosensing, drug delivery, synthetic biology, and molecular computation fields, where precise control over the transduction of binding events into a specific output is key.

Silver oxide model surface improves computational simulation of surface-enhanced Raman spectroscopy on silver nanoparticles.

Surface-enhanced Raman spectroscopy (SERS) coupled with density functional theory (DFT) computations can characterise the adsorption orientation of a molecule on a nanoparticle surface. When using DFT to simulate SERS on a silver surface, one typically employs an atom (Ag), ion (Ag+), or cluster (Agx or Agx+) as the model surface. Here, by examining the nucleobase 2,6-diaminopurine (2,6-DAP) and then generalising our strategy to three other molecules, we show that employing silver oxide (Ag2O) as the model surface can quantitatively improve the accuracy of simulated SERS.

Peptide-Mediated Electrochemical Steric Hindrance Assay for One-Step Detection of HIV Antibodies.

Diagnosis of infectious disease in patients, including human immunodeficiency virus (HIV) infection, can be achieved through the detection of specific antibodies produced by the immune system. We have previously shown that macromolecules such as antibodies can be efficiently detected in complex biological samples by sterically inhibiting the hybridization of conjugated complementary DNA strands to electrode-bound DNA strands. Here, we report a peptide-mediated electrochemical steric hindrance hybridization assay, PeSHHA, specially for the detection of antibodies against the gp41 protein of HIV-1. We show that the sensitivity of this PeSHHA can be significantly enhanced using nanostructured electrodes and demonstrate the rapid, one-step detection of HIV-1 antibodies directly in clinical samples.

Determining the folding and binding free energy of DNA-based nanodevices and nanoswitches using urea titration curves.

DNA nanotechnology takes advantage of the predictability of DNA interactions to build complex DNA-based functional nanoscale structures. However, when DNA functional and responsive units that are based on non-canonical DNA interactions are employed it becomes quite challenging to predict, understand and control their thermodynamics. In response to this limitation, here we demonstrate the use of isothermal urea titration experiments to estimate the free energy involved in a set of DNA-based systems ranging from unimolecular DNA-based nanoswitches to more complex DNA folds (e.g. aptamers) and nanodevices. We propose here a set of fitting equations that allow to analyze the urea titration curves of these DNA responsive units based on Watson-Crick and non-canonical interactions (stem-loop, G-quadruplex, triplex structures) and to correctly estimate their relative folding and binding free energy values under different experimental conditions. The results described herein will pave the way toward the use of urea titration experiments in the field of DNA nanotechnology to achieve easier and more reliable thermodynamic characterization of DNA-based functional responsive units. More generally, our results will be of general utility to characterize other complex supramolecular systems based on different biopolymers.

Engineering Biosensors with Dual Programmable Dynamic Ranges.

Although extensively used in all fields of chemistry, molecular recognition still suffers from a significant limitation: host-guest binding displays a fixed, hyperbolic dose-response curve, which limits its usefulness in many applications. Here we take advantage of the high programmability of DNA chemistry and propose a universal strategy to engineer biorecognition-based sensors with dual programmable dynamic ranges. Using DNA aptamers as our model recognition element and electrochemistry as our readout signal, we first designed a dual signaling "signal-on" and "signal-off" adenosine triphosphate (ATP) sensor composed of a ferrocene-labeled ATP aptamer in complex to a complementary, electrode-bound, methylene-blue labeled DNA. Using this simple "dimeric" sensor, we show that we can easily (1) tune the dynamic range of this dual-signaling sensor through base mutations on the electrode-bound DNA, (2) extend the dynamic range of this sensor by 2 orders of magnitude by using a combination of electrode-bound strands with varying affinity for the aptamers, (3) create an ultrasensitive dual signaling sensor by employing a sequestration strategy in which a nonsignaling, high affinity "depletant" DNA aptamer is added to the sensor surface, and (4) engineer a sensor that simultaneously provides extended and ultrasensitive readouts. These strategies, applicable to a wide range of biosensors and chemical systems, should broaden the application of molecular recognition in various fields of chemistry.

A DNA Nanodevice That Loads and Releases a Cargo with Hemoglobin-Like Allosteric Control and Cooperativity.

Here we report the rational design of a synthetic molecular nanodevice that is directly inspired from hemoglobin, a highly evolved protein whose oxygen-carrying activity is finely regulated by a sophisticated network of control mechanisms. Inspired by the impressive performance of hemoglobin we have designed and engineered in vitro a synthetic DNA-based nanodevice containing up to four interacting binding sites that, like hemoglobin, can load and release a cargo over narrow concentration ranges, and whose affinity can be finely controlled via both allosteric effectors and environmental cues like pH and temperature. As the first example of a synthetic DNA nanodevice that undergoes a complex network of nature-inspired control mechanisms, this represents an important step toward the use of similar nanodevices for diagnostic and drug-delivery applications.

Biomolecular Steric Hindrance Effects Are Enhanced on Nanostructured Microelectrodes.

The availability of rapid approaches for quantitative detection of biomarkers would drastically impact global health by enabling decentralized disease diagnosis anywhere that patient care is administered. A promising new approach, the electrochemical steric hindrance hybridization assay (eSHHA) has been introduced for quantitative detection of large proteins (e.g., antibodies) with a low nanomolar detection limit within 10 min. Here, we report the use of a nanostructured microelectrode (NME) platform for eSHHA that improves the performance of this approach by increasing the efficiency and kinetics of DNA hybridization. We demonstrated that eSHHA on nanostructured microelectrodes leverages three effects: (1) steric hindrance effects at the nanoscale, (2) a size-dependent hybridization rate of DNA complexes, and (3) electrode morphology-dependent blocking effects. As a proof of concept, we showed that the sensitivity of eSHHA toward a model antibody is enhanced using NMEs as scaffolds for this reaction. We improved the detection limit of eSHHA, taking advantage of nanostructured surfaces to allow the use of longer capture strands for detection of proteins. Finally, we concluded that using the eSHHA approach in conjunction with nanostructured microelectrodes is an advantageous alternative to conventional macroelectrodes as the sensitivity and detection limits are enhanced.

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.

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.

Programmable Quantitative DNA Nanothermometers.

Developing molecules, switches, probes or nanomaterials that are able to respond to specific temperature changes should prove of utility for several applications in nanotechnology. Here, we describe bioinspired strategies to design DNA thermoswitches with programmable linear response ranges that can provide either a precise ultrasensitive response over a desired, small temperature interval (±0.05 °C) or an extended linear response over a wide temperature range (e.g., from 25 to 90 °C). Using structural modifications or inexpensive DNA stabilizers, we show that we can tune the transition midpoints of DNA thermometers from 30 to 85 °C. Using multimeric switch architectures, we are able to create ultrasensitive thermometers that display large quantitative fluorescence gains within small temperature variation (e.g., > 700% over 10 °C). Using a combination of thermoswitches of different stabilities or a mix of stabilizers of various strengths, we can create extended thermometers that respond linearly up to 50 °C in temperature range. Here, we demonstrate the reversibility, robustness, and efficiency of these programmable DNA thermometers by monitoring temperature change inside individual wells during polymerase chain reactions. We discuss the potential applications of these programmable DNA thermoswitches in various nanotechnology fields including cell imaging, nanofluidics, nanomedecine, nanoelectronics, nanomaterial, and synthetic biology.

General Strategy to Introduce pH-Induced Allostery in DNA-Based Receptors to Achieve Controlled Release of Ligands.

Inspired by naturally occurring pH-regulated receptors, here we propose a rational approach to introduce pH-induced allostery into a wide range of DNA-based receptors. To demonstrate this we re-engineered two model DNA-based probes, a molecular beacon and a cocaine-binding aptamer, by introducing in their sequence a pH-dependent domain. We demonstrate here that we can finely tune the affinity of these model receptors and control the load/release of their specific target molecule by a simple pH change.

Enzyme-Operated DNA-Based Nanodevices.

Functional molecular nanodevices and nanomachines have attracted a growing interest for their potential use in life science and nanomedicine. In particular, due to their versatility and modularity DNA-based nanodevices appear extremely promising. However, a limitation of such devices is represented by the limited number of molecular stimuli and cues that can be used to control and regulate their function. Here we demonstrate the possibility to rationally control and regulate DNA-based nanodevices using biocatalytic reactions catalyzed by different enzymes. To demonstrate the versatility of our approach, we have employed three model DNA-based systems and three different enzymes (belonging to several classes, i.e., transferases and hydrolases). The possibility to use enzymes and enzymatic substrates as possible cues to operate DNA-based molecular nanodevices will expand the available toolbox of molecular stimuli to be used in the field of DNA nanotechnology and could open the door to many applications including enzyme-induced drug delivery and enzyme-triggered nanostructures assembly.

Controlling Hybridization Chain Reactions with pH.

By taking inspiration from nature, where self-organization of biomolecular species into complex systems is finely controlled through different stimuli, we propose here a rational approach by which the assembly and disassembly of DNA-based concatemers can be controlled through pH changes. To do so we used the hybridization chain reaction (HCR), a process that, upon the addition of an initiator strand, allows to create DNA-based concatemers in a controlled fashion. We re-engineered the functional units of HCR through the addition of pH-dependent clamp-like triplex-forming domains that can either inhibit or activate the polymerization reaction at different pHs. This allows to finely regulate the HCR-induced assembly and disassembly of DNA concatemers at either basic or acidic pHs in a reversible way. The strategies we present here appear particularly promising as novel tools to achieve better spatiotemporal control of self-assembly processes of DNA-based nanostructures.

A Highly Selective Electrochemical DNA-Based Sensor That Employs Steric Hindrance Effects to Detect Proteins Directly in Whole Blood.

Here we describe a highly selective DNA-based electrochemical sensor that utilizes steric hindrance effects to signal the presence of large macromolecules in a single-step procedure. We first show that a large macromolecule, such as a protein, when bound to a signaling DNA strand generates steric hindrance effects, which limits the ability of this DNA to hybridize to a surface-attached complementary strand. We demonstrate that the efficiency of hybridization of this signaling DNA is inversely correlated with the size of the molecule attached to it, following a semilogarithmic relationship. Using this steric hindrance hybridization assay in an electrochemical format (eSHHA), we demonstrate the multiplexed, quantitative, one-step detection of various macromolecules in the low nanomolar range, in <10 min directly in whole blood. We discuss the potential applications of this novel signaling mechanism in the field of point-of-care diagnostic sensors.

A Modular, DNA-Based Beacon for Single-Step Fluorescence Detection of Antibodies and Other Proteins.

A versatile platform for the one-step fluorescence detection of both monovalent and multivalent proteins has been developed. This system is based on a conformation-switching stem-loop DNA scaffold that presents a small-molecule, polypeptide, or nucleic-acid recognition element on each of its two stem strands. The steric strain associated with the binding of one (multivalent) or two (monovalent) target molecules to these elements opens the stem, enhancing the emission of an attached fluorophore/quencher pair. The sensors respond rapidly (<10 min) and selectively, enabling the facile detection of specific proteins even in complex samples, such as blood serum. The versatility of the platform was demonstrated by detecting five bivalent proteins (four antibodies and the chemokine platelet-derived growth factor) and two monovalent proteins (a Fab fragment and the transcription factor TBP) with low nanomolar detection limits and no detectable cross-reactivity.

Programmable pH-triggered DNA nanoswitches.

We have designed programmable DNA-based nanoswitches whose closing/opening can be triggered over specific different pH windows. These nanoswitches form an intramolecular triplex DNA structure through pH-sensitive parallel Hoogsteen interactions. We demonstrate that by simply changing the relative content of TAT/CGC triplets in the switches, we can rationally tune their pH dependence over more than 5 pH units. The ability to design DNA-based switches with tunable pH dependence provides the opportunity to engineer pH nanosensors with unprecedented wide sensitivity to pH changes. For example, by mixing in the same solution three switches with different pH sensitivity, we developed a pH nanosensor that can precisely monitor pH variations over 5.5 units of pH. With their fast response time (<200 ms) and high reversibility, these pH-triggered nanoswitches appear particularly suitable for applications ranging from the real-time monitoring of pH changes in vivo to the development of pH sensitive smart nanomaterials.

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.