Oleg Lioubashevski

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.

Ultrasensitive Surface Plasmon Resonance Detection of Trinitrotoluene by a Bis-aniline-Cross-Linked Au Nanoparticles Composite

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.

Au-Nanoparticle Nanowires Based on DNA and Polylysine Templates

The assembly of ordered nanoparticle architectures is a challenging topic in nanotechnology directed to the construction of nanoscale devices.[1] Within this broad subject, the conjugation of biomaterials and nanoparticles to yield ordered architectures is a promising route to tailor future sensing and catalytic devices, nanocircuitry, or nanodevices, for example transistors, and computing devices.[2] DNA is an attractive biomaterial for use as a template in programmed nanoparticle structures. The ability to synthesize nucleic acids of predesigned shapes and composition, the versatile biocatalytic transformations that can be performed on DNA, for example, ligation, scission, or polymerization, enable TMcut and paste∫ procedures to be carried out on the template DNA, thus enabling us to design and manipulate the DNA ∫mold∫. Furthermore, the association of metal ions to the DNA phosphate units, or the intercalation of transition-metal complexes or molecular substrates into the DNA provide a means to functionalize the DNA-template and to initiate further chemical transformations on the mold. Nanoparticle ± DNA assemblies were organized by the hybridization of nucleic-acid-functionalized metal[3] or semiconductor nanoThe fluorescence data in our experiments indicated that the surface coverage of the final printed layer for each of the three patterning methods presented here is nearly equivalent and reaches about 60% of the surface coverage obtained by direct deposition of the antibodies from solution. As already described for CP and CP of proteins, the printing process does not compromise the binding efficiency of the printed antibody. This strategy might not be suitable for patterning a large number of different proteins on a surface. However, it can place a few different proteins as adjacent high-density arrays on a surface. Such arrays could find an application for high-throughput screening in which a large number of analytes could be spotted using a subset of the patterned areas. Another possibility for creating high-density immunoassays on planar surfaces is by performing surface immunoassays using many different analytes and capture sites, such as shown in Figure 5. The main limiting factor in using the prepared microarrays for diagnostic purposes could be misplacement of target molecules during the inking of the stamp. Such a misplacement, which may induce false positive reactions, can arise from cross-reactions of the target molecules with different capture proteins and/or from nonspecific adsorption on the -stamp. The former is limited by biological specificity of affinity extraction. The latter can be limited by the systematic use of blocking agents such as BSA. Indeed, for the recognition of goat antigen by the printed array shown in Figure 5a, the recognition signal in the areas with printed anti-chicken antibodies was only 5% of that in the areas with printed anti-goat antibodies. In summary, we have illustrated how CP can complement different patterning methods to produce repeatedly, and in parallel, high resolution arrays of proteins in three simple steps: 1) TMinking∫, 2) rinsing, and 3) printing the stamp on the substrate. Since -stamps carry the complementary pattern of binding partners specific to the target proteins on their surface, the proteins self-assemble into the predefined array on the stamp surface during inking in solution, and dissociate upon printing. Hence, the (re)production of the target protein arrays is fast and easy. The initial production of the -stamp is a one-time burden only. We thus believe that the methodology presented is powerful and versatile, and should be useful in detection and fabrication strategies that are based on arrays of proteins.

Concatenated logic gates using four coupled biocatalysts operating in series

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.

Two coupled enzymes perform in parallel the ‘AND’ and ‘InhibAND’ logic gate operations

The coupled activation of two enzymes: glucose dehydrogenase (GDH) and horseradish peroxidase (HRP), is used to construct the parallel-operating AND and InhibAND logic gates. The added substrates for the respective enzymes, glucose and H(2)O(2), act as the gate inputs, while the biocatalytically generated NADH and gluconic acid provide the output signals that follow the operations of the gates. The two gates are generated in the same vial, thus allowing the logic operations to take place in parallel, and the simultaneous readout of the functions of the gates.

A full-adder based on reconfigurable DNA-hairpin inputs and DNAzyme computing modules

In nature, post-transcriptional alternative splicing processes expand the proteome biodiversity, providing means to synthesize various protein isoforms. We describe the input-guided assembly of a DNAzyme-based full-adder computing system, which mimics functions of the natural processes by increasing the diversity of logic elements by the reconfiguration of the inputs. The full-adder comprises the simultaneous operation of three inputs that yield two different output signals, acting as sum and carry bits. The DNAzyme-based full-adder system consists of a library of Mg2+-dependent DNAzyme subunits and their substrates that are modified by two different fluorophore/quencher pairs that encode the sum and carry outputs. The input-guided assembly of DNAzyme subunits, formed by three inputs composed of nucleic acid hairpin structures, leads to computing modules that yield the sum and carry outputs of the full-adder. In the presence of a single input the DNAzyme computing module yields the sum fluorescence output. In the presence of two of the inputs, the reconfiguration of the input structures proceeds, leading to an input-guided computing module that yields the carry fluorescence output. By introducing all the three inputs the sequential inter-input hybridization leads to the reconfiguration of the inputs into polymer wires. These include binding sites for two types of DNAzyme and their substrates leading to the carry and sum fluorescence outputs. The advantages of the simultaneous three-input operation of the full-adder and the possibilities to implement DNAzyme-based computing modules for cascading full-adders are discussed.

Self-assembly of luminescent Ag nanocluster-functionalized nanowires.

Two different methods to self-assemble red- or yellow-luminescent nucleic acids-stabilized Ag nanoclusters (NCs) nanowires are presented. By one method, the autonomous hybridization-polymerization process between two nucleic acids leads to polymer chains consisting of sequence-specific loops for the stabilization of the red- or yellow-emitting Ag NCs. By the other method, the nucleic acid-triggered hybridization chain reaction (HCR) involving the cross-opening of two functional hairpins leads to sequence-specific DNA loops and a nucleic acid scaffold that stabilize the respective red- or yellow-emitting Ag NCs. The micrometer-long luminescent Ag NC-functionalized nanowires are imaged by AFM and confocal microscopy.

Rewriting the Story of Excimer Formation in Liquid Benzene

Formation of benzene excimer following UV excitation of the neat liquid is monitored with femtosecond spectroscopy. A prompt rise component in excimer transient absorption, which contradicts the classical scenario of gradual reorientation and pairing of the excited monomers, is observed. Three-pulse experiments in which the population of evolving excimers is depleted by a secondary dump pulse demonstrate that the excimer absorption band is polarized along the interfragment axis. The experiments furthermore prove that the subsequent 4-fold increase in excimer absorption over ∼50 ps is primarily due to an increase in the transition dipole of pairs which are formed early on, and not to excited monomers forming excimers in a delayed fashion due to unfavorable initial geometry. Results are analyzed in light of recent studies of local structure in the liquid benzene combined with advanced electronic structure calculations. The prompt absorption rise is ascribed to excited states delocalized over nearby benzene molecules, which are sufficiently close and nearly parallel in the pure liquid. Such low-symmetry structures, which differ considerably from the optimized structures of isolated benzene dimer and solid benzene, are sufficiently abundant in liquid benzene. Electronic structure calculations confirm the orientation of transition dipoles of the excimers along the interparticle axis and demonstrate how slow refinement of the intermolecular geometry leads to a significant increase in the excimer absorption strength.

Magneto-switchable single-electron charging of Au-nanoparticles using hydrophobic magnetic nanoparticles

Reversible magneto-switchable quantum charging of a Au nanoparticle array associated with a Au electrode is observed in the presence of hydrophobic magnetic nanoparticles attracted to the functionalized electrode surface.

Nonresonant Raman Effects on Femtosecond Pump–Probe with Chirped White Light: Challenges and Opportunities

Impulsive Raman excitation in neat organic liquids far from resonance is followed using chirped broad-band supercontinuum probe pulses. Spectral modulations due to impulsively induced coherent vibrations vary in intensity 10-fold as a function of the probes linear chirp. Simulations clarify why the vibrational signature is maximized for a group delay dispersion (GDD) in reduced units of νvib-2 = 0.5 while a probe GDD of twice that quenches the same spectral modulations. Accordingly, recent claims that chirped white-light probe pulses provide equivalent information on material response to their compressed analogues must be taken with caution. In particular, interactions that induce spectral shifts in the probe depend crucially on the arrival chronology of the continuum colors. On one hand, this presents limitations to application of chirped continuum radiation as-is in pump-probe experiments. It also presents the opportunity for using this dependence to control the relative amplitude of nonresonant interactions in pump-probe signals such as that of solvent vibrations.