The ability of DNA to self-assemble into one-, two- and three-dimensional nanostructures, combined with the precision that is now possible when positioning nanoparticles or proteins on DNA scaffolds, provide a promising approach for the self-organization of composite nanostructures. Predicting and controlling the functions that emerge in self-organized biomolecular nanostructures is a major challenge in systems biology, and although a number of innovative examples have been reported, the emergent properties of systems in which enzymes are coupled together have not been fully explored. Here, we report the self-assembly of a DNA scaffold made of DNA strips that include 'hinges' to which biomolecules can be tethered. We attach either two enzymes or a cofactor-enzyme pair to the scaffold, and show that enzyme cascades or cofactor-mediated biocatalysis can proceed effectively; similar processes are not observed in diffusion-controlled homogeneous mixtures of the same components. Furthermore, because the relative position of the two enzymes or the cofactor-enzyme pair is determined by the topology of the DNA scaffold, it is possible to control the reactivity of the system through the design of the individual DNA strips. This method could lead to the self-organization of complex multi-enzyme cascades.
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.
Different selected enzymes, glucose oxidase (GOx), catalase (Cat), glucose dehydrogenase (GDH), horseradish peroxidase (HRP), and formaldehyde dehydrogenase (FDH), are used alone or coupled to construct eight different logic gates. The added substrates for the respective enzymes, glucose and H(2)O(2), act as the gate inputs, while the biocatalytically generated gluconic acid or NADH are the output signals that follow the operation of the gates. Different enzyme-based gates are XOR, INHIBIT A, INHIBIT B, AND, OR, NOR, Identity and Inverter gates. By combining the AND and XOR or the XOR and INHIBIT A gates, the half-adder and half-subtractor are constructed, respectively, opening the way to elementary computing by the use of enzymes.
The assembly of three concatenated enzyme-based logic gates consisting of OR, AND, XOR is described. Four biocatalysts, acetylcholine esterase, choline oxidase, microperoxidase-11, and the NAD ؉ -dependent glucose dehydrogenase, are used to assemble the gates. Four inputs that include acetylcholine, butyrylcholine, O2, and glucose are used to drive the concatenated-gates system. The cofactor NAD ؉ , and its reduced 1,4-dihydro form, NADH, are used as a reporter couple, and these provide an optical output for the gates. The modulus of the absorbance changes of NADH is used as a readout signal.biocomputers ͉ biocatalysis ͉ enzymes T he hardware of computers consists of parallel and serial logic-gate operations that are triggered by electronic inputs. These functions may be duplicated by appropriately designed chemical or biological systems. Different molecular and supramolecular assemblies that operate as logic gates and perform molecular-scale arithmetic operations were discussed (1-9). Similarly, biomolecules such as nucleic acids or proteins were used as active components that perform logic-gate operations (10-15). Gene-based artificial circuits acting as bistable toggle switches (16) or oscillators (17) were developed, and coupled enzyme͞DNA systems that perform programmable biochemical transformations that mimic basic computing of finite automaton were reported (17). The use of enzymes as the active components for logic gate functions is specifically intriguing because numerous biocatalytic cycles in nature rely on information processing, revealing similarities to computer devices. Although the function of enzyme networks as mimics of Boolean logic gates was discussed (18), and the potential use of enzymes as building units of high-density computing architectures was addressed (19), the experimental work that validates biocatalyst-stimulated logic gate operations is quite scarce, and lacks the desired complexity that resembles computers. Single enzyme-based logic-gate operations were reported. For example, the dynamic conformational changes of malate dehydrogenase in response to Mg ϩ and Ca 2ϩ ions acting as inputs was used to develop a XOR gate (20). Also, a modified enzyme and its inhibitor were used as inputs that activated an AND gate (21). Recently, we have reported on the assembly of coupled biocatalytic systems that mimic OR, XOR, AND, or InhibAND logic-gate functions (22), and the use of these systems for elementary arithmetic operations (halfadder and half-subtractor) was demonstrated (23). In none of these systems was the consecutive operation of several gates that operate in series demonstrated. This feature is, however, essential to develop any future ''computer-like'' function of enhanced complexity. Here, we report on the assembly of a four-enzymecoupled system that includes four inputs and performs in series three logic-gate operations OR, AND, and XOR. Results and DiscussionsThe system and its operation is depicted in Fig. 1A and consists of the four biocatalysts, acetylcholine esterase (AChE)...
A bis-aniline-cross-linked Au nanoparticles (NPs) composite is electropolymerized on Au surfaces. The association of trinitrotoluene, TNT, to the bis-aniline bridging units via pi-donor-acceptor interactions allows the amplified detection of TNT by following the surface plasmon resonance (SPR) reflectance changes as a result of the coupling between the localized plasmon of the AuNPs and the surface plasmon wave associated with the gold surface. The detection limit for analyzing TNT by this method is approximately 10 pM. The electropolymerization of the bis-aniline-cross-linked AuNPs composite in the presence of picric acid results in a molecular-imprinted matrix for the enhanced binding of TNT. The imprinted AuNPs composite enabled the sensing of TNT with a detection limit that corresponded to 10 fM. Analysis of the SPR reflectance changes in the presence of different concentrations of TNT revealed a two-step calibration curve that included the ultrasensitive detection of TNT by the imprinted sites in the composite, KassI. for the association of TNT to the imprinted sites, 6.4 x 10(12) M-1, followed by a less sensitive detection of TNT by the nonimprinted pi-donor bis-aniline sites (KNIass. = 3.9 x 10(9) M-1). The imprinted AuNPs composite reveals impressive selectivity. The structural and functional features of the bis-aniline-cross-linked AuNPs composites were characterized by different methods including ellipsometry, AFM, and electrochemical means. The dielectric properties of the AuNPs composite in the presence of different concentrations of TNT were evaluated by the theoretical fitting of the respective experimental SPR curves. The ultrasensitive detection of the TNT by the AuNPs composite was attributed to the changes of the dielectric properties of the composite, as a result of the formation of the pi-donor-acceptor complexes between TNT and the bis-aniline units. These changes in the dielectric properties lead to a change in the conductivity of the AuNPs matrix.
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