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

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

Engineering Biosensors with Extended, Narrowed, or Arbitrarily Edited Dynamic Range

Biomolecular recognition has long been an important theme in artificial sensing technologies. A current limitation of protein- and nucleic acid-based recognition, however, is that the useful dynamic range of single-site binding typically spans an 81-fold change in target concentration, an effect that limits the utility of biosensors in applications calling for either great sensitivity (a steeper relationship between target concentration and output signal) or the quantification of more wide-ranging concentrations. In response, we have adapted strategies employed by nature to modulate the input–output response of its biorecognition systems to rationally edit the useful dynamic range of an artificial biosensor. By engineering a structure-switching mechanism to tune the affinity of a receptor molecule, we first generated a set of receptor variants displaying similar specificities but different target affinities. Using combinations of these receptor variants (signaling and nonsignaling), we then rationally extended (to 900000-fold), narrowed (to 5-fold), and edited (three-state) the normally 81-fold dynamic range of a representative biosensor. We believe that these strategies may be widely applicable to technologies reliant on biorecognition

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

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

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

Thermodynamic Basis for Engineering High-Affinity, High-Specificity Binding-Induced DNA Clamp Nanoswitches

Naturally occurring chemoreceptors almost invariably employ structure-switching mechanisms, an observation that has inspired the use of biomolecular switches in a wide range of artificial technologies in the areas of diagnostics, imaging, and synthetic biology. In one mechanism for generating such behavior, clamp-based switching, binding occurs <i>via</i> the clamplike embrace of two recognition elements onto a single target molecule. In addition to coupling recognition with a large conformational change, this mechanism offers a second advantage: it improves both affinity and specificity simultaneously. To explore the physics of such switches we have dissected here the thermodynamics of a clamp-switch that recognizes a target DNA sequence through both Watson–Crick base pairing and triplex-forming Hoogsteen interactions. When compared to the equivalent linear DNA probe (which relies solely on Watson–Crick interactions), the extra Hoogsteen interactions in the DNA clamp-switch increase the probe’s affinity for its target by ∼0.29 ± 0.02 kcal/mol/base. The Hoogsteen interactions of the clamp-switch likewise provide an additional specificity check that increases the discrimination efficiency toward a single-base mismatch by 1.2 ± 0.2 kcal/mol. This, in turn, leads to a 10-fold improvement in the width of the “specificity window” of this probe relative to that of the equivalent linear probe. Given these attributes, clamp-switches should be of utility not only for sensing applications but also, in the specific field of DNA nanotechnology, for applications calling for a better control over the building of nanostructures and nanomachines

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

Electrochemical DNA-Based Immunoassay That Employs Steric Hindrance To Detect Small Molecules Directly in Whole Blood

The development of a universal sensing mechanism for the rapid and quantitative detection of small molecules directly in whole blood would drastically impact global health by enabling disease diagnostics, monitoring, and treatment at home. We have previously shown that hybridization between a free DNA strand and its complementary surface-bound strand can be sterically hindered when the former is bound to large antibodies. Here, we exploit this effect to design a competitive antibody-based electrochemical assay, called CeSHHA, that enables the quantitative detection of small molecules directly in complex matrices, such as whole blood or soil. We discuss the importance of this inexpensive assay for point-of-care diagnosis and for treatment monitoring applications

Allosterically Tunable, DNA-Based Switches Triggered by Heavy Metals

Here we demonstrate the rational design of allosterically controllable, metal-ion-triggered molecular switches. Specifically, we designed DNA sequences that adopt two low energy conformations, one of which does not bind to the target ion and the other of which contains mismatch sites serving as specific recognition elements for mercury­(II) or silver­(I) ions. Both switches contain multiple metal binding sites and thus exhibit <i>homotropic allosteric</i> (cooperative) responses. As <i>heterotropic</i> allosteric effectors we employ single-stranded DNA sequences that either stabilize or destabilize the nonbinding state, enabling dynamic range tuning over several orders of magnitude. The ability to rationally introduce these effects into target-responsive switches could be of value in improving the functionality of DNA-based nanomachines