Andrea Idili

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.

Rational Design of pH-Controlled DNA Strand Displacement

Achieving strategies to finely regulate with biological inputs the formation and functionality of DNA-based nanoarchitectures and nanomachines is essential toward a full realization of the potential of DNA nanotechnology. Here we demonstrate an unprecedented, rational approach to achieve control, through a simple change of the solutions pH, over an important class of DNA association-based reactions. To do so we took advantage of the pH dependence of parallel Hoogsteen interactions and rationally designed two triplex-based DNA strand displacement strategies that can be triggered and finely regulated at either basic or acidic pHs. Because pH change represents an important input both in healthy and pathological biological pathways, our findings can have implication for the development of DNA nanostructures whose assembly and functionality can be triggered in the presence of specific biological targets.

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.

Folding-Upon-Binding and Signal-On Electrochemical DNA Sensor with High Affinity and Specificity

Here we investigate a novel signal-on electrochemical DNA sensor based on the use of a clamp-like DNA probe that binds a complementary target sequence through two distinct and sequential events, which lead to the formation of a triplex DNA structure. We demonstrate that this target-binding mechanism can improve both the affinity and specificity of recognition as opposed to classic probes solely based on Watson–Crick recognition. By using electrochemical signaling to report the conformational change, we demonstrate a signal-on E-DNA sensor with up to 400% signal gain upon target binding. Moreover, we were able to detect with nanomolar affinity a perfectly matched target as short as 10 bases (KD = 0.39 nM). Finally, thanks to the molecular “double-check” provided by the concomitant Watson–Crick and Hoogsteen base pairings involved in target recognition, our sensor provides excellent discrimination efficiency toward a single-base mismatched target.

Selective control of reconfigurable chiral plasmonic metamolecules

DNA origami nanotechnology enables selective reconfiguration of plasmonic quasi-enantiomers through simple pH tuning. Selective configuration control of plasmonic nanostructures using either top-down or bottom-up approaches has remained challenging in the field of active plasmonics. We demonstrate the realization of DNA-assembled reconfigurable plasmonic metamolecules, which can respond to a wide range of pH changes in a programmable manner. This programmability allows for selective reconfiguration of different plasmonic metamolecule species coexisting in solution through simple pH tuning. This approach enables discrimination of chiral plasmonic quasi-enantiomers and arbitrary tuning of chiroptical effects with unprecedented degrees of freedom. Our work outlines a new blueprint for implementation of advanced active plasmonic systems, in which individual structural species can be programmed to perform multiple tasks and functions in response to independent external stimuli.

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.

Antibody-powered nucleic acid release using a DNA-based nanomachine

A wide range of molecular devices with nanoscale dimensions have been recently designed to perform a variety of functions in response to specific molecular inputs. Only limited examples, however, utilize antibodies as regulatory inputs. In response to this, here we report the rational design of a modular DNA-based nanomachine that can reversibly load and release a molecular cargo on binding to a specific antibody. We show here that, by using three different antigens (including one relevant to HIV), it is possible to design different DNA nanomachines regulated by their targeting antibody in a rapid, versatile and highly specific manner. The antibody-powered DNA nanomachines we have developed here may thus be useful in applications like controlled drug-release, point-of-care diagnostics and in vivo imaging.

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.

Simulative and Experimental Characterization of a pH-Dependent Clamp-like DNA Triple-Helix Nanoswitch

Here we couple experimental and simulative techniques to characterize the structural/dynamical behavior of a pH-triggered switching mechanism based on the formation of a parallel DNA triple helix. Fluorescent data demonstrate the ability of this structure to reversibly switch between two states upon pH changes. Two accelerated, half microsecond, MD simulations of the system having protonated or unprotonated cytosines, mimicking the pH 5.0 and 8.0 conditions, highlight the importance of the Hoogsteen interactions in stabilizing the system, finely depicting the time-dependent disruption of the hydrogen bond network. Urea-unfolding experiments and MM/GBSA calculations converge in indicating a stabilization energy at pH 5.0, 2-fold higher than that observed at pH 8.0. These results validate the pH-controlled behavior of the designed structure and suggest that simulative approaches can be successfully coupled with experimental data to characterize responsive DNA-based nanodevices.

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

Abstract 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.