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.e., urea and H2O2). To demonstrate the programmability of such DNA-based swimmers, we also engineer DNA strands that displace the enzyme from the DNA scaffold, thus acting as molecular "brakes" on the DNA swimmers. These results serve as a first proof of principle for the development of synthetic DNA-based enzyme-powered swimmers that can self-propel in fluids.

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. We show that by controlling the degradation rate of blocker and input strands, we can finely control the delayed pulse output over a range of 10 h. We also prove that it is possible to orthogonally delay two different pulse reactions in the same solution by taking advantage of the specificity of the degradation reactions for the input and blocker strands. Finally, we show here two possible applications of such delayed pulse SDRs: the time-programmed pulse decoration of DNA nanostructures and the sequentially appearing and self-erasing formation of DNA-based patterns.

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. That is, even if an "as-selected" aptamer achieves useful affinity and specificity, it may fail when adapted to the EAB platform because it lacks the binding-induced conformational change required to support EAB signaling. In this paper we reveal the spectroscopy-guided approaches we use to modify aptamers such that they support the necessary binding-induced conformational change. Specifically, using newly reported aptamers, we demonstrate the systematic design of EAB sensors achieving clinically and physiologically relevant specificity, limits of detection, and dynamic range against the targets methotrexate and tryptophan.

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. The concentration of the disulfide fuels determines the activation kinetics of each DNA tile, which controls the degree of order/disorder in the formed co-polymer. The disulfide-reduction pathway can be employed together with enzymatic fuel-degradation pathways providing an additional level of control in the re-organization of DNA structures. Taking advantage of the different pH-sensitivities of disulfide-thiol and enzymatic reactions, we show that we can control the order in DNA-based co-polymers as a function of pH.

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. antigens, aptamers or proteins) and with orthogonal fluorescent dyes. As a proof-of-principle we have developed sandwich and competitive multiplex lateral flow platforms for the detection of several targets, ranging from small molecules (digoxigenin, Dig and dinitrophenol, DNP), to antibodies (Anti-Dig, Anti-DNP and Anti-MUC1/EGFR bispecific antibodies) and proteins (thrombin). Coupling the advantages of functional DNA-based scaffolds together with the simplicity of LFAs, our approach offers the opportunity to detect a wide range of targets with nanomolar sensitivity and high specificity.

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. The kinetics of the blocker enzymatic degradation thus controls the time at which the SDR starts. By varying the concentration of the blocker strand and the concentration of the enzyme, we show that we can finely tune and modulate the delayed onset of SDR. Additionally, we show that the strategy is versatile and can be orthogonally controlled by different enzymes each specifically targeting a different blocker strand. We designed and established three different delayed SDRs using RNase H and two DNA repair enzymes (formamidopyrimidine DNA glycosylase and uracil-DNA glycosylase) and corresponding blockers. The achieved temporal delay can be programed with high flexibility without undesired leak and can be conveniently predicted using kinetic modeling. Finally, we show three possible applications of the delayed SDRs to temporally control the ligand release from a DNA nanodevice, the inhibition of a target protein by a DNA aptamer, and the output signal generated by a DNA logic circuit.

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. The approach is programmable, specific and orthogonal and the two responsive modules can be used in the same solution without crosstalk. This allows to assemble structures formed by two different tiles in which the tile distribution can be accurately predicted as a function of the relative activity of each enzyme. Finally, we show that BER-enzyme inhibitors can also be used to control DNA-tile assembly in a specific and concentration-dependent manner.

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. We show here two possible applications of this strategy: the transient labelling of DNA structures and the additional temporal control of cascade reactions.

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. We measure the dynamic reconfiguration via fluorescent signals and confocal microscopy, and we derive a kinetic model that captures the experimental results. Our approach suggests a novel route toward the development of biomolecular materials in which engineered chemical reactions support the autonomous spatial reorganization of multiple components.

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. We show that by sequentially adding specific inputs it is possible to re-organize the formed structures to display well-defined distributions: homopolymers, random and block structures. The versatility of the systems presented in this study shows the ease with which DNA-based addressable monomers can be designed to create reconfigurable micron-scale DNA structures offering a new approach to the growing field of supramolecular polymers.

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. Upon reduction, the allosteric modulator spontaneously de-hybridizes from the nanodevice and, as a result, its effect is transient. The system is reversible and has an unprecedented high tolerance to waste products and displays transient behavior for over 40 cycles without significant loss of efficiency. Kinetic control of DNA-based ligand-binding nanodevices through purely chemical reactions paves the way for temporal regulation of more complex chemical pathways.

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. Although highly efficient, enzymes pose limits in terms of long-term stability and inhibition of enzyme activity by waste products. Herein, we provide the first example of kinetically controlled DNA nanostructures in which energy dissipation is achieved through a non-enzymatic chemical reaction. More specifically, inspired by redox signalling, we employ redox cycles of disulfide-bond formation/breakage to kinetically control the assembly and disassembly of tubular DNA nanostructures in a highly controllable and reversible fashion.

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. We achieved protein-controlled regulation of a synthetic RNA and of an enzyme through artificial DNA-based communication, showing the potential of our molecular system in performing further programmable tasks.

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. When RNA nanotechnology meets bioanalytical chemistry, sensing of target molecules can be performed by harnessing programmable interactions of RNA modules, advanced field-ready biosensors can be manufactured by interfacing RNA-based devices with supporting portable platforms, and RNA sensors can be engineered to be genetically encoded allowing for real-time imaging of biomolecules in living cells. In this article, we report recent advances in RNA-based sensing technologies and discuss current trends in RNA nanotechnology-enabled biomedical diagnostics. In particular, we describe programmable sensors that leverage modular designs comprising dynamic aptamer-based units, synthetic RNA nanodevices able to perform target-responsive regulation of gene expression, and paper-based sensors incorporating artificial RNA networks. Graphical Abstract ᅟ.

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. We demonstrate that this effect is specific and versatile and allows the orthogonal control of multiple reactions and multiple structures in the same solution. Moreover, the strategy is highly tunable and can be finely modulated by varying the cathodic potential, the period of applied potential, and the density of the DNA strand on the chip surface. Our approach thus represents a versatile way to remotely control DNA-based circuits and nanostructure assembly and can allow new possible applications of DNA-based nanotools.

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. The use of an enzyme that specifically cleaves the inhibitor only when it is bound to the aptamer generates a transient allosteric control that leads to the release of ATP or cocaine from the aptamers. Our approach confirms that the programmability and predictability of nucleic acids make synthetic DNA/RNA the perfect candidate material to re-engineer synthetic receptors that can undergo chemical fuel-triggered release of small-molecule cargoes and to rationally design non-equilibrium systems.

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. Finally, it is shown that chemical fuels can be used to selectively activate different functions in a system of higher complexity embedded with multiple response pathways.

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.

The impact of nitric oxide toxicity on the evolution of the glutathione transferase superfamily: a proposal for an evolutionary driving force.

Glutathione transferases (GSTs) are protection enzymes capable of conjugating glutathione (GSH) to toxic compounds. During evolution an important catalytic cysteine residue involved in GSH activation was replaced by serine or, more recently, by tyrosine. The utility of these replacements represents an enigma because they yield no improvements in the affinity toward GSH or in its reactivity. Here we show that these changes better protect the cell from nitric oxide (NO) insults. In fact the dinitrosyl·diglutathionyl·iron complex (DNDGIC), which is formed spontaneously when NO enters the cell, is highly toxic when free in solution but completely harmless when bound to GSTs. By examining 42 different GSTs we discovered that only the more recently evolved Tyr-based GSTs display enough affinity for DNDGIC (KD < 10(-9) M) to sequester the complex efficiently. Ser-based GSTs and Cys-based GSTs show affinities 10(2)-10(4) times lower, not sufficient for this purpose. The NO sensitivity of bacteria that express only Cys-based GSTs could be related to the low or null affinity of their GSTs for DNDGIC. GSTs with the highest affinity (Tyr-based GSTs) are also over-represented in the perinuclear region of mammalian cells, possibly for nucleus protection. On the basis of these results we propose that GST evolution in higher organisms could be linked to the defense against NO.

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.e., Ribonuclease H, RNase H, or Uracil DNA Glycosylase, UDG) inducing their spontaneous de-hybridization from the assembled tile and replacement by a new strand conjugated to a different label. Multiple enzyme-responsive strands that specifically respond to different enzymes allow for dynamic, orthogonal, and reversible decoration of the DNA structures. As a proof-of-principle of the strategy, the possibility to orthogonally control the distribution of different labels (i.e., fluorophores and small molecules) on the same scaffold without crosstalk is demonstrated. By doing so, DNA scaffolds that display different antibody recognition patterns are obtained. The approach offers the possibility to control the decoration of higher-order supramolecular assemblies (including origami) with several functional moieties to achieve functional biomaterials with improved adaptability, precision, and sensing capabilities.