Enzyme-Responsive DNA Condensates.

Membrane-less compartments and organelles are widely acknowledged for their role in regulating cellular processes, and there is an urgent need to harness their full potential as both structural and functional elements of synthetic cells. Despite rapid progress, synthetically recapitulating the nonequilibrium, spatially distributed responses of natural membrane-less organelles remains elusive. Here, we demonstrate that the activity of nucleic-acid cleaving enzymes can be localized within DNA-based membrane-less compartments by sequestering the respective DNA or RNA substrates.

Synthetic DNA-based Swimmers Driven by Enzyme Catalysis.

Here, we report DNA-based synthetic nanostructures decorated with enzymes (hereafter referred to as DNA-enzyme swimmers) that self-propel by converting the enzymatic substrate to the product in solution. The DNA-enzyme swimmers are obtained from tubular DNA structures that self-assemble spontaneously by the hybridization of DNA tiles. We functionalize these DNA structures with two different enzymes, urease and catalase, and show that they exhibit concentration-dependent movement and enhanced diffusion upon addition of the enzymatic substrate (i.

Decorated DNA-Based Scaffolds as Lateral Flow Biosensors.

Here we develop Lateral Flow Assays (LFAs) that employ as functional elements DNA-based structures decorated with reporter tags and recognition elements. We have rationally re-engineered tile-based DNA tubular structures that can act as scaffolds and can be decorated with recognition elements of different nature (i.e.

Timed Pulses in DNA Strand Displacement Reactions.

Inspired by naturally occurring regulatory mechanisms that allow complex temporal pulse features with programmable delays, we demonstrate here a strategy to achieve temporally programmed pulse output signals in DNA-based strand displacement reactions (SDRs). To achieve this, we rationally designed input strands that, once bound to their target duplex, can be gradually degraded, resulting in a pulse output signal. We also designed blocker strands that suppress strand displacement and determine the time at which the pulse reaction is generated.

Autonomous and Programmable Reorganization of DNA-Based Polymers Using Redox Chemistry.

We demonstrate here a strategy that allows the programmable and autonomous reorganization of self-assembled DNA polymers using redox chemistry. We have rationally designed different DNA monomers (tiles) that can co-assemble into tubular structures. The tiles can be orthogonally activated/deactivated with disulfide-linked DNA fuel strands that are degraded over time upon reduction because of the presence of a reducing agent in the system.

Dynamic and Reversible Decoration of DNA-Based Scaffolds.

An approach to achieving dynamic and reversible decoration of DNA-based scaffolds is demonstrated here. To do this, rationally engineered DNA tiles containing enzyme-responsive strands covalently conjugated to different molecular labels are employed. These strands are designed to be recognized and degraded by specific enzymes (i.

Controlling DNA nanodevices with light-switchable buffers.

Control over synthetic DNA-based nanodevices can be achieved with a variety of physical and chemical stimuli. Actuation with light, however, is as advantageous as difficult to implement without modifying DNA strands with photo-switchable groups. Herein, we show that DNA nanodevices can be controlled using visible light in photo-switchable aqueous buffer solutions in a reversible and highly programmable fashion.

Orthogonal Enzyme-Driven Timers for DNA Strand Displacement Reactions.

Here, we demonstrate a strategy to rationally program a delayed onset of toehold-mediated DNA strand displacement reactions (SDRs). The approach is based on blocker strands that efficiently inhibit the strand displacement by binding to the toehold domain of the target DNA. Specific enzymatic degradation of the blocker strand subsequently enables SDR.

Using Spectroscopy to Guide the Adaptation of Aptamers into Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based (EAB) sensors utilize the binding-induced conformational change of an electrode-attached, redox-reporter-modified aptamer to transduce target recognition into an easily measurable electrochemical output. Because this signal transduction mechanism is single-step and rapidly reversible, EAB sensors support high-frequency, real-time molecular measurements, and because it recapitulates the reagentless, conformation-linked signaling seen in vivo among naturally occurring receptors, EAB sensors are selective enough to work in the complex, time-varying environments found in the living body. The fabrication of EAB sensors, however, requires that their target-recognizing aptamer be modified such that (1) it undergoes the necessary binding-induced conformational change and (2) that the thermodynamics of this "conformational switch" are tuned to ensure that they reflect an acceptable trade-off between affinity and signal gain.

DNA Tile Self-Assembly Guided by Base Excision Repair Enzymes.

We demonstrate here the use of DNA repair enzymes to control the assembly of DNA-based structures. To do so, we employed uracil-DNA glycosylase (UDG) and formamidopyrimidine DNA glycosylase (Fpg), two enzymes involved in the base excision repair (BER) pathway. We designed two responsive nucleic acid modules containing mutated bases (deoxyuridine or 8-oxo-7,8-dihydroguanine recognized by UDG and Fpg, respectively) that, upon the enzyme repair activity, release a nucleic acid strand that induces the self-assembly of DNA tiles into tubular structures.

Dissipative DNA nanotechnology.

DNA nanotechnology has emerged as a powerful tool to precisely design and control molecular circuits, machines and nanostructures. A major goal in this field is to build devices with life-like properties, such as directional motion, transport, communication and adaptation. Here we provide an overview of the nascent field of dissipative DNA nanotechnology, which aims at developing life-like systems by combining programmable nucleic-acid reactions with energy-dissipating processes.

Dissipative Control over the Toehold-Mediated DNA Strand Displacement Reaction.

Here we show a general approach to achieve dissipative control over toehold-mediated strand-displacement, the most widely employed reaction in the field of DNA nanotechnology. The approach relies on rationally re-engineering the classic strand displacement reaction such that the high-energy invader strand (fuel) is converted into a low-energy waste product through an energy-dissipating reaction allowing the spontaneous return to the original state over time. We show that such dissipative control over the toehold-mediated strand displacement process is reversible (up to 10 cycles), highly controllable and enables unique temporal activation of DNA systems.

Spontaneous Reorganization of DNA-Based Polymers in Higher Ordered Structures Fueled by RNA.

We demonstrate a strategy that allows for the spontaneous reconfiguration of self-assembled DNA polymers exploiting RNA as chemical fuel. To do this, we have rationally designed orthogonally addressable DNA building blocks that can be transiently deactivated by RNA fuels and subtracted temporarily from participation in the self-assembly process. Through a fine modulation of the rate at which the building blocks are reactivated we can carefully control the final composition of the polymer and convert a disordered polymer in a higher order polymer, which is disfavored from a thermodynamic point of view.

Reorganization of Self-Assembled DNA-Based Polymers using Orthogonally Addressable Building Blocks*.

Nature uses non-covalent interactions to achieve structural dynamic reconfiguration of biopolymers. Taking advantage of the programmability of DNA/DNA interactions we report here the rational design of orthogonal DNA-based addressable tiles that self-assemble into polymer-like structures that can be reconfigured by external inputs. The different tiles share the same sticky ends responsible for self-assembly but are rationally designed to contain a specific regulator-binding domain that can be orthogonally targeted by different DNA regulator strands.

Disulfide-Linked Allosteric Modulators for Multi-cycle Kinetic Control of DNA-Based Nanodevices.

Nature employs sulfur switches, that is, redox-active disulfides, to kinetically control biological pathways in a highly efficient and reversible way. Inspired by this mechanism, we describe herein a DNA-based synthetic nanodevice that acts as a sulfur switch and can be temporally controlled though redox regulation. To do this, we rationally designed disulfide DNA strands (modulators) that hybridize to a ligand-binding DNA nanodevice and act as redox-active allosteric regulators inducing the nanodevice to release or load its ligand.

Protein-Controlled Actuation of Dynamic Nucleic Acid Networks by Using Synthetic DNA Translators*.

Integrating dynamic DNA nanotechnology with protein-controlled actuation will expand our ability to process molecular information. We have developed a strategy to actuate strand displacement reactions using DNA-binding proteins by engineering synthetic DNA translators that convert specific protein-binding events into trigger inputs through a programmed conformational change. We have constructed synthetic DNA networks responsive to two different DNA-binding proteins, TATA-binding protein and Myc-Max, and demonstrated multi-input activation of strand displacement reactions.

Transient DNA-Based Nanostructures Controlled by Redox Inputs.

Synthetic DNA has emerged as a powerful self-assembled material for the engineering of nanoscale supramolecular devices and materials. Recently dissipative self-assembly of DNA-based supramolecular structures has emerged as a novel approach providing access to a new class of kinetically controlled DNA materials with unprecedented life-like properties. So far, dissipative control has been achieved using DNA-recognizing enzymes as energy dissipating units.

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.

Programmable RNA-based systems for sensing and diagnostic applications.

The emerging field of RNA nanotechnology harnesses the versatility of RNA molecules to generate nature-inspired systems with programmable structure and functionality. Such methodology has therefore gained appeal in the fields of biosensing and diagnostics, where specific molecular recognition and advanced input/output processing are demanded. The use of RNA modules and components allows for achieving diversity in structure and function, for processing information with molecular precision, and for programming dynamic operations on the grounds of predictable non-covalent interactions.

Fuel-Responsive Allosteric DNA-Based Aptamers for the Transient Release of ATP and Cocaine.

We show herein that allostery offers a key strategy for the design of out-of-equilibrium systems by engineering allosteric DNA-based nanodevices for the transient loading and release of small organic molecules. To demonstrate the generality of our approach, we used two model DNA-based aptamers that bind ATP and cocaine through a target-induced conformational change. We re-engineered these aptamers so that their affinity towards their specific target is controlled by a DNA sequence acting as an allosteric inhibitor.

Remote Electronic Control of DNA-Based Reactions and Nanostructure Assembly.

The use of synthetic DNA to design and build molecular machines and well-defined structures at the nanoscale has greatly impacted the field of nanotechnology. Here we expand the current toolkit in this field by demonstrating an efficient, quantitative, and versatile approach that allows us to remotely control DNA-based reactions and DNA nanostructure self-assembly using electronic inputs. To do so we have deposited onto the surface of disposable chips different DNA input strands that upon the application of a cathodic potential can be desorbed in a remote and controlled way and trigger DNA-based reactions and DNA nanostructure self-assembly.

Dissipative Synthetic DNA-Based Receptors for the Transient Loading and Release of Molecular Cargo.

Supramolecular chemistry is moving into a direction in which the composition of a chemical equilibrium is no longer determined by thermodynamics but by the efficiency with which kinetic states can be populated by energy consuming processes. Herein, we show that DNA is ideally suited for programming chemically fueled dissipative self-assembly processes. Advantages of the DNA-based systems presented in this study include a perfect control over the activation site for the chemical fuel in terms of selectivity and affinity, highly selective fuel consumption that occurs exclusively in the activated complex, and a high tolerance for the presence of waste products.

A modular clamp-like mechanism to regulate the activity of nucleic-acid target-responsive nanoswitches with external activators.

Here we demonstrate a general and modular approach to regulate the activity of target-responsive DNA-based nanoswitches. We do so by coupling together two DNA-based responsive elements: a triplex-forming clamp-like probe able to bind a specific DNA sequence and a split aptamer selected to bind a small molecule. In the presence of the specific target of one of the above responsive elements, the nanoswitch partially folds and its ability to bind the second target is restored.

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.