Johann Elbaz

DNA computing circuits using libraries of DNAzyme subunits

Biological systems that are capable of performing computational operations could be of use in bioengineering and nanomedicine, and DNA and other biomolecules have already been used as active components in biocomputational circuits. There have also been demonstrations of DNA/RNA-enzyme-based automatons, logic control of gene expression, and RNA systems for processing of intracellular information. However, for biocomputational circuits to be useful for applications it will be necessary to develop a library of computing elements, to demonstrate the modular coupling of these elements, and to demonstrate that this approach is scalable. Here, we report the construction of a DNA-based computational platform that uses a library of catalytic nucleic acids (DNAzymes), and their substrates, for the input-guided dynamic assembly of a universal set of logic gates and a half-adder/half-subtractor system. We demonstrate multilayered gate cascades, fan-out gates and parallel logic gate operations. In response to input markers, the system can regulate the controlled expression of anti-sense molecules, or aptamers, that act as inhibitors for enzymes.

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.

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.

A DNAzyme cascade for the amplified detection of Pb2+ ions or L-histidine

DNAzyme cascades activated by Pb(2+)- or L-histidine-dependent DNAzymes yield the horseradish peroxidase-mimicking catalytic nucleic acids that enable the colorimetric or chemiluminescence detection of Pb(2+) or L-histidine.

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.

Powering the programmed nanostructure and function of gold nanoparticles with catenated DNA machines

DNA nanotechnology is a rapidly developing research area in nanoscience. It includes the development of DNA machines, tailoring of DNA nanostructures, application of DNA nanostructures for computing, and more. Different DNA machines were reported in the past and DNA-guided assembly of nanoparticles represents an active research effort in DNA nanotechnology. Several DNA-dictated nanoparticle structures were reported, including a tetrahedron, a triangle or linear nanoengineered nanoparticle structures; however, the programmed, dynamic reversible switching of nanoparticle structures and, particularly, the dictated switchable functions emerging from the nanostructures, are missing elements in DNA nanotechnology. Here we introduce DNA catenane systems (interlocked DNA rings) as molecular DNA machines for the programmed, reversible and switchable arrangement of different-sized gold nanoparticles. We further demonstrate that the machine-powered gold nanoparticle structures reveal unique emerging switchable spectroscopic features, such as plasmonic coupling or surface-enhanced fluorescence.

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.

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.

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.

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.