Programmable On-Chip Artificial Cell Producing Post-Translationally Modified Ubiquitinated Protein.

In nature, intracellular microcompartments have evolved to allow the simultaneous execution of tightly regulated complex processes within a controlled environment. This architecture serves as the blueprint for the construction of a wide array of artificial cells. However, such systems are inadequate in their ability to confine and sequentially control multiple central dogma activities (transcription, translation, and post-translational modifications) resulting in a limited production of complex biomolecules. Here, an artificial cell-on-a-chip comprising hierarchical compartments allowing the processing and transport of products from transcription, translation, and post-translational modifications through connecting channels is designed and fabricated. This platform generates a tightly controlled system, yielding directly a purified modified protein, with the potential to produce proteoform of choice. Using this platform, the full ubiquitinated form of the Parkinson's disease-associated α-synuclein is generated starting from DNA, in a single device. By bringing together all central dogma activities in a single controllable platform, this approach will open up new possibilities for the synthesis of complex targets, will allow to decipher diverse molecular mechanisms in health and disease and to engineer protein-based materials and pharmaceutical agents.

Au nanoparticle/DNA rotaxane hybrid nanostructures exhibiting switchable fluorescence properties.

The preparation of a DNA rotaxane consisting of a circular nucleic acid interlocked, through hybridization, on a nucleic acid axle and stoppered by two 10-nm-sized Au nanoparticles (NPs) is described. By the tethering of 5-nm- or 15-nm-sized Au NPs on the ring, the supramolecular structure of the rotaxane is confirmed. Using nucleic acids as "fuels" and "anti-fuels", the cyclic and reversible transition of the rotaxane ring across two states is demonstrated. By the functionalization of the ring with fluorophore-modified nucleic acids in different orientations, the transitions of the rings between the sites are followed by fluorescence quenching or surface-enhanced fluorescence. The experimental results are supported by theoretical modeling.

A three-station DNA catenane rotary motor with controlled directionality.

The assembly of DNA machines represents a central effort in DNA nanotechnology. We report on the first DNA rotor system composed of a two-ring catenane. The DNA rotor ring rotates in dictated directions along a wheel, and it occupies three distinct sites. Hg(2+)/cysteine or pH (H(+)/OH(-)) act as fuels or antifuels in positioning the rotor ring. Analysis of the kinetics reveals directional clockwise or anticlockwise population of the target-sites (>85%), and the rotor's direction is controlled by the shortest path on the wheel.

All-DNA finite-state automata with finite memory.

Biomolecular logic devices can be applied for sensing and nano-medicine. We built three DNA tweezers that are activated by the inputs H(+)/OH(-); ; nucleic acid linker/complementary antilinker to yield a 16-states finite-state automaton. The outputs of the automata are the configuration of the respective tweezers (opened or closed) determined by observing fluorescence from a fluorophore/quencher pair at the end of the arms of the tweezers. The system exhibits a memory because each current state and output depend not only on the source configuration but also on past states and inputs.

pH-programmable DNA logic arrays powered by modular DNAzyme libraries.

Nature performs complex information processing circuits, such the programmed transformations of versatile stem cells into targeted functional cells. Man-made molecular circuits are, however, unable to mimic such sophisticated biomachineries. To reach these goals, it is essential to construct programmable modular components that can be triggered by environmental stimuli to perform different logic circuits. We report on the unprecedented design of artificial pH-programmable DNA logic arrays, constructed by modular libraries of Mg(2+)- and UO(2)(2+)-dependent DNAzyme subunits and their substrates. By the appropriate modular design of the DNA computation units, pH-programmable logic arrays of various complexities are realized, and the arrays can be erased, reused, and/or reprogrammed. Such systems may be implemented in the near future for nanomedical applications by pH-controlled regulation of cellular functions or may be used to control biotransformations stimulated by bacteria.

Enzyme-free amplified detection of DNA by an autonomous ligation DNAzyme machinery.

The Zn(2+)-dependent ligation DNAzyme is implemented as a biocatalyst for the amplified detection of a target DNA by the autonomous replication of a nucleic acid reporter unit that is generated by the catalyzed ligation process. The reporter units enhance the formation of active DNAzyme units, thus leading to the isothermal autocatalytic formation of the reporter elements. The system was further developed and applied for the amplified detection of Tay-Sachs genetic disorder mutant, with a detection limit of 1.0 × 10(-11) M. Besides providing a versatile paradigm for the amplified detection of DNA, the system reveals a new, enzyme-free, isothermal, autocatalytic mechanism that introduces means for effective programmed synthesis.

DNA machines: bipedal walker and stepper.

The assembly of a "bipedal walker" and of a "bipedal stepper" using DNA constructs is described. These DNA machines are activated by H(+)/OH(-) and Hg(2+)/cysteine triggers. The bipedal walker is activated on a DNA template consisting of four nucleic acid footholds. The forward "walking" of the DNA on the template track is activated by Hg(2+) ions and H(+) ions, respectively, using the thymine-Hg(2+)-thymine complex or the i-motif structure as the DNA translocation driving forces. The backward "walking" is activated by OH(-) ions and cysteine, triggers that destroy the i-motif or thymine-Hg(2+)-thymine complexes. Similarly, the "bipedal stepper" is activated on a circular DNA template consisting of four tethered footholds. With the Hg(2+)/cysteine and H(+)/OH(-) triggers, clockwise or anticlockwise stepping is demonstrated. The operation of the DNA machines is followed optically by the appropriate labeling of the walker-foothold components with the respective fluorophores/quenchers units.

Parallel engineering of environmental bacteria and performance over years under jungle-simulated conditions.

Engineered bacteria could perform many functions in the environment, for example, to remediate pollutants, deliver nutrients to crops or act as in-field biosensors. Model organisms can be unreliable in the field, but selecting an isolate from the thousands that naturally live there and genetically manipulating them to carry the desired function is a slow and uninformed process. Here, we demonstrate the parallel engineering of isolates from environmental samples by using the broad-host-range XPORT conjugation system (Bacillus subtilis mini-ICEBs1) to transfer a genetic payload to many isolates in parallel. Bacillus and Lysinibacillus species were obtained from seven soil and water samples from different locations in Israel. XPORT successfully transferred a genetic function (reporter expression) into 25 of these isolates. They were then screened to identify the best-performing chassis based on the expression level, doubling time, functional stability in soil, and environmentally-relevant traits of its closest annotated reference species, such as the ability to sporulate and temperature tolerance. From this library, we selected Bacillus frigoritolerans A3E1, re-introduced it to soil, and measured function and genetic stability in a contained environment that replicates jungle conditions. After 21 months of storage, the engineered bacteria were viable, could perform their function, and did not accumulate disruptive mutations.

Amplified analysis of DNA by the autonomous assembly of polymers consisting of DNAzyme wires.

A systematic study of the amplified optical detection of DNA by Mg(2+)-dependent DNAzyme subunits is described. The use of two DNAzyme subunits and the respective fluorophore/quencher-modified substrate allows the detection of the target DNA with a sensitivity corresponding to 1 × 10(-9) M. The use of two functional hairpin structures that include the DNAzyme subunits in a caged, inactive configuration leads, in the presence of the target DNA, to the opening of one of the hairpins and to the activation of an autonomous cross-opening process of the two hairpins, which affords polymer DNA wires consisting of the Mg(2+)-dependent DNAzyme subunits. This amplification paradigm leads to the analysis of the target DNA with a sensitivity corresponding to 1 × 10(-14) M. The amplification mixture composed of the two hairpins can be implemented as a versatile sensing platform for analyzing any gene in the presence of the appropriate hairpin probe. This is exemplified with the detection of the BRCA1 oncogene.

Genetically Encoding Ultrastable Virus-like Particles Encapsulating Functional DNA Nanostructures in Living Bacteria.

DNA nanotechnology is leading the field of in vitro molecular-scale device engineering, accumulating to a dazzling array of applications. However, while DNA nanostructures' function is robust under in vitro settings, their implementation in real-world conditions requires overcoming their rapid degradation and subsequent loss of function. Viruses are sophisticated supramolecular assemblies, able to protect their nucleic acid content in inhospitable biological environments. Inspired by this natural ability, we engineered in vitro and in vivo technologies, enabling the encapsulation and protection of functional DNA nanostructures inside MS2 bacteriophage virus-like particles (VLPs). We demonstrate the ssDNA-VLPs nanocomposites' (NCs) abilities to encapsulate single-stranded-DNA (ssDNA) in a variety of sizes (200-1500 nucleotides (nt)), sequences, and structures while retaining their functionality. Moreover, by exposing these NCs to hostile biological conditions, such as human blood serum, we exhibit that the VLPs serve as an excellent protective shell. These engineered NCs pose critical properties that are yet unattainable by current fabrication methods.

Electrochemically stimulated pH changes: a route to control chemical reactivity.

A bis-aniline-cross-linked Au nanoparticle (NP) composite is electrochemically prepared on a rough Pt film supported on a Au electrode. The electrochemical oxidation of the bis-aniline units to the quinoid state releases protons to the electrolyte solution, while the reduction of the quinoid bridges results in the uptake of protons from the electrolyte. By the cyclic oxidation of the bridging units (E = 0.25 V vs SCE), and their reduction (E = -0.05 V vs SCE), the pH of the solution could be reversibly switched between the values 5.8 and 7.2, respectively. The extent of the pH change is controlled by the number of electropolymerization cycles applied to synthesize the Au NP composite, demonstrating a ca. 1.5 pH units change by a matrix synthesized using 100 electropolymerization cycles. The pH changes are used to reversibly activate and deactivate a C-quadruplex (i-motif)-bridged Mg(2+)-dependent DNAzyme.

Nanoengineered electrically contacted enzymes on DNA scaffolds: functional assemblies for the selective analysis of Hg2+ ions.

A DNA construct consisting of a nucleic acid template, (1), on which a nucleic acid-modified glucose oxidase (GOx), (3), was hybridized by cooperative bridging of the T-Hg(2+)-T units, and a nucleic acid-functionalized ferrocene, (5), was directly hybridized on a Au electrode. The resulting nanostructure revealed bioelectrocatalytic activities, where the ferrocene units mediated electron transfer between the redox center of the enzyme and the electrode. The bioelectrocatalytic functions of the system are regulated by the concentration of Hg(2+) ions, which controls the content of the enzyme associated with the DNA template by means of the T-Hg(2+)-T bridging units. This phenomenon allowed the amperometric detection of Hg(2+) ions at a detection limit 1 x 10(-10) M with impressive selectivity.

Amplified biosensing using the horseradish peroxidase-mimicking DNAzyme as an electrocatalyst.

The hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme is assembled on Au electrodes. It reveals bioelectrocatalytic properties and electrocatalyzes the reduction of H(2)O(2). The bioelectrocatalytic functions of the hemin/G-quadruplex DNAzyme are used to develop electrochemical sensors that follow the activity of glucose oxidase and biosensors for the detection of DNA or low-molecular-weight substrates (adenosine monophosphate, AMP). Hairpin nucleic structures that include the G-quadruplex sequence in a caged configuration and the nucleic acid sequence complementary to the analyte DNA, or the aptamer sequence for AMP, are immobilized on Au-electrode surfaces. In the presence of the DNA analyte, or AMP, the hairpin structures are opened, and the hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme structures are generated on the electrode surfaces. The bioelectrocatalytic cathodic currents generated by the functionalized electrodes, upon the electrochemical reduction of H(2)O(2), provide a quantitative measure for the detection of the target analytes. The DNA target was analyzed with a detection limit of 1 x 10(-12) M, while the detection limit for analyzing AMP was 1 x 10(-6) M. Methods to regenerate the sensing surfaces are presented.

pH-stimulated concurrent mechanical activation of two DNA "tweezers". A "SET-RESET" logic gate system.

A DNA tweezer consisting of C-rich arms is kept in the "closed" form by hybridization of the arms with a nucleic acid cross-linker. At acidic pH (pH = 5.2), the arms are stabilized through the formation of the i-motif, C-quadruplex structures, releasing the cross-linking nucleic acid and transforming the tweezer to its "opened" state. At neutral pH (pH = 7.2), the C-quadruplex structures are dissociated, resulting in the capturing of the cross-linking nucleic acid and the closure of the tweezer. By the reversible treatment of the tweezer at pH = 5.2 and at pH = 7.2, the tweezer system is cycled between the open and closed states, respectively, followed by a FRET process between a fluorophore-quencher pair that labels the tweezer. Also the concurrent activation of two DNA tweezers by pH stimuli is described. The pH-induced opening of one tweezer (tweezer A) by the formation of C-quadruplex (pH = 5.2) and the release of the cross-linking nucleic acid result in the closure of a second tweezer (tweezer B) by the hybridization of the released strand with the arms of tweezer B. The dissociation of the C-quadruplex structures (pH = 7.2) results in the favored translocation of the cross-linking nucleic acid from tweezer B to A. By the cycling of the pH of the system between pH = 5.2 and pH = 7.2, the concurrent opening and closure of the two tweezers are accomplished. The two tweezers system performs a SET-RESET logic gate operation, where the pH stimuli act as inputs.

Engineering a DNAzyme-Based Operon System for the Production of DNA Nanoscaffolds in Living Bacteria.

The ability to create nanoscaffolds within living cells using DNA has the potential to become a powerful tool in synthetic biology. However, to date, genetically encoded DNA nanostructures are limited to simple architecture due to the lack of genetic parts that can produce multiple ssDNAs in a single bacterium. Here, we develop a system that overcomes this challenge by using a single oligo gene mimicking operons. This was achieved by converting a noncoding RNA into a long ssDNA that self-cleaves into multiple ssDNAs using R3-DNAzymes (DNAzyme-based operon). We demonstrate the ability to apply the DNAzyme-based operon to produce a four-ssDNA crossover nanostructure (25 nm) that recruits split YFPs when properly assembled. This system enables the formation of more complex DNA nanostructures in vivo and thus paves the way to further integrate the field of DNA nanotechnology into living bacteria for basic biology, bioengineering, and medicine applications.

Cooperative multicomponent self-assembly of nucleic acid structures for the activation of DNAzyme cascades: a paradigm for DNA sensors and aptasensors.

The activation of a DNAzyme cascade by the cooperative self-assembly of multicomponent nucleic acid structures is suggested as a method for the amplified sensing of DNA, or the specific substrates of aptamers. According to one configuration, the DNA analyte 1 is detected by two tailored nucleic acids 2 and 3 that form a multicomponent supramolecular structure with a ribonucleobase-containing quasi-circular DNA 4, but only upon the concomitant hybridization with 1. The resulting supramolecular nucleic acid structure includes the Mg(2+)-dependent DNAzyme that cleaves the ribonucleobase site of 4. The cleavage of the quasi-circular DNA 4 results in the fragmentation of the supramolecular structure and the release of two horseradish peroxidase (HRP) mimicking units that were incorporated in the blocked quasi-circular DNA 4. The HRP-mimicking DNAzyme catalyzed the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS(2-)) by H(2)O(2) to ABTS(*-), and the product provided the colorimetric readout signal for the analyzed DNA. The method enabled the analysis of DNA with a detection limit of 1 x 10(-12) M. Similarly, an analogous DNAzyme cascade was activated by the low-molecular-weight substrates, adenosine triphosphate (ATP) or cocaine. This was induced by the self-assembly of nucleic acids that included fragments of the respective aptamers and the Mg(2+)-dependent DNAzyme. Furthermore, nucleic acids consisting of fragments of the aptamers against ATP or cocaine and fragments of the HRP-mimicking DNAzyme self-assemble, in the presence of the respective substrates, to the active DNAzyme structure that catalyzes the oxidation of ABTS(2-) by H(2)O(2) to form the colored product ABTS(*-). The resulting product provided the readout signal for the recognition events. The cooperative interaction in the formation of the supramolecular nucleic acid assemblies and the activation of the DNAzymes are discussed.

Self-assembly of supramolecular aptamer structures for optical or electrochemical sensing.

The self-assembly of labeled aptamer sub-units in the presence of their substrates provides a method for the optical (fluorescence) or electrochemical detection of the substrate. One of the sub-units is linked to CdSe/ZnS quantum dots (QDs), and the self-assembly of the dye-functionalized second sub-unit with the modified QDs, in the presence of cocaine, stimulates fluorescence resonance energy transfer (FRET). This enables the detection of cocaine with a detection limit corresponding to 1 x 10(-6) M. Alternatively, the aptamer fragments are modified with pyrene units. The formation of a supramolecular aptamer-substrate complex allosterically stabilizes the formation of excimer supramolecular structure, and its characteristic emission is observed. In addition, the thiolated aptamer sub-unit is assembled on an Au electrode. The Methylene Blue-labeled sub-unit binds to the surface-confined fragment in the presence of cocaine. The amperometric response of the system allows the detection of cocaine with a detection limit of 1 x 10(-5) M. The approach is generic and can be applied to other substrates, e.g. adenosine triphosphate.

Coherent activation of DNA tweezers: a "SET-RESET" logic system.

Open sesame: Aptamer-substrate complexes activate the coherent operation of two tweezers that act as a "SET-RESET" logic system. Each tweezer cycles between a fluorescent open state and a closed quenched state (Q = quencher, F = fluorophore) when triggered by adenosine monophosphate (AMP) and adenosine deaminase (AD).

Parallel analysis of two analytes in solutions or on surfaces by using a bifunctional aptamer: applications for biosensing and logic gate operations.

A bifunctional aptamer that includes two aptamer units for cocaine and adenosine 5'-monophosphate (AMP) is blocked by a nucleic acid to form a hybrid structure with two duplex regions. The blocked bifunctional aptamer assembly is used as a functional structure for the simultaneous sensing of cocaine or AMP. The blocked bifunctional aptamer is dissociated by either of the two analytes, and the readout of the separation of the sensing structure is accomplished by a colorimetric detection, by a released DNAzyme, or by electronic means that use Faradaic impedance spectroscopy or field-effect transistors. In one configuration, the blocked bifunctional aptamer structure is separated by the substrates cocaine or AMP, and the displaced blocker units act as a horseradish peroxidase-mimicking DNAzyme that permits the colorimetric detection of the analytes. In the second system, the blocked bifunctional aptamer hybrid is associated with a Au electrode. The displacement of the aptamer by any of the substrates alters the interfacial electron transfer resistance at the electrode surface, thus providing an electronic signal for the sensing process. In the third configuration, the blocked aptamer hybrid is linked to the gate of a field-effect transistor device. The separation of the complex by means of any of the analytes, cocaine, or AMP alters the gate potential, and this allows the electronic transduction of the sensing process by following the changes in the gate-to-source potentials. The different systems enable not only the simultaneous detection of the two analytes, but they provide a functional assembly that performs a logic gate "OR" operation.