Ron Orbach

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.

Self-Assembled Fmoc-Peptides as a Platform for the Formation of Nanostructures and Hydrogels

Hydrogels are of great interest as a class of materials for tissue engineering, axonal regeneration, and controlled drug delivery, as they offer 3D interwoven scaffolds to support the growth of cells. Herein, we extend the family of the aromatic Fmoc-dipeptides with a library of new Fmoc-peptides, which include natural and synthetic amino acids with an aromatic nature. We describe the self-assembly of these Fmoc-peptides into various structures and characterize their distinctive molecular and physical properties. Moreover, we describe the fabrication of the bioactive RGD sequence into a hydrogel. This unique material offers new opportunities for developing cell-adhesive biomedical hydrogel scaffolds, as well as for establishing strategies to modify surfaces with bioactive materials.

pH‐Stimulated DNA Hydrogels Exhibiting Shape‐Memory Properties

Nucleic acid-functionalized polyacrylamide chains that are cooperatively cross-linked by i-motif and nucleic acid duplex units yield, at pH 5.0, DNA hydrogels exhibiting shape-memory properties. Separation of the i-motif units at pH 8.0 dissolves the hydrogel into a quasi-liquid phase. The residual duplex units provide, however, a memory code in the quasi-liquid allowing the regeneration of the hydrogel shape at pH 5.0.

The rheological and structural properties of Fmoc-peptide-based hydrogels: the effect of aromatic molecular architecture on self-assembly and physical characteristics.

Biocompatible hydrogels are of high interest as a class of biomaterials for tissue engineering, regenerative medicine, and controlled drug delivery. These materials offer three-dimensional scaffolds to support the growth of cells and development of hierarchical tissue structures. Fmoc-peptides were previously demonstrated as attractive building blocks for biocompatible hydrogels. Here, we further investigate the biophysical properties of Fmoc-peptide-based hydrogels for medical applications. We describe the structural and thermal properties of these Fmoc-peptides, as well as their self-assembly process. Additionally, we study the role of interactions between aromatic moieties in the self-assembly process and on the physical and structural properties of the hydrogels.

Logic reversibility and thermodynamic irreversibility demonstrated by DNAzyme-based Toffoli and Fredkin logic gates

The Toffoli and Fredkin gates were suggested as a means to exhibit logic reversibility and thereby reduce energy dissipation associated with logic operations in dense computing circuits. We present a construction of the logically reversible Toffoli and Fredkin gates by implementing a library of predesigned Mg2+-dependent DNAzymes and their respective substrates. Although the logical reversibility, for which each set of inputs uniquely correlates to a set of outputs, is demonstrated, the systems manifest thermodynamic irreversibility originating from two quite distinct and nonrelated phenomena. (i) The physical readout of the gates is by fluorescence that depletes the population of the final state of the machine. This irreversible, heat-releasing process is needed for the generation of the output. (ii) The DNAzyme-powered logic gates are made to operate at a finite rate by invoking downhill energy-releasing processes. Even though the three bits of Toffoli’s and Fredkin’s logically reversible gates manifest thermodynamic irreversibility, we suggest that these gates could have important practical implication in future nanomedicine.

Switchable Bifunctional Stimuli-Triggered Poly-N-Isopropylacrylamide/DNA Hydrogels†

DNA-tethered poly-N-isopropylacrylamide copolymer chains, pNIPAM, that include nucleic acid tethers have been synthesized. They are capable of inducing pH-stimulated crosslinking of the chains by i-motif structures or to be bridged by Ag(+) ions to form duplexes. The solutions of pNIPAM chains undergo crosslinking at pH 5.2 or in the presence of Ag(+) ions to form hydrogels. The hydrogels reveal switchable hydrogel-to-solution transitions by the reversible crosslinking of the chains at pH 5.2 and the separation of the crosslinking units at pH 7.5, or by the Ag(+) ion-stimulated crosslinking of the chains and the reverse dissolution of the hydrogel by the cysteamine-induced elimination of the Ag(+) ions. The DNA-crosslinked hydrogels are thermosensitive and undergo reversible temperature-controlled hydrogel-to-solid transitions. The solid pNIPAM matrices are protected against the OH(-) or cysteamine-stimulated dissociation to the respective polymer solutions.

Self-assembly of DNA nanotubes with controllable diameters

The synthesis of DNA nanotubes is an important area in nanobiotechnology. Different methods to assemble DNA nanotubes have been reported, and control over the width of the nanotubes has been achieved by programmed subunits of DNA tiles. Here we report the self-assembly of DNA nanotubes with controllable diameters. The DNA nanotubes are formed by the self-organization of single-stranded DNAs, exhibiting appropriate complementarities that yield hexagon (small or large) and tetragon geometries. In the presence of rolling circle amplification strands, that exhibit partial complementarities to the edges of the hexagon- or tetragon-building units, non-bundled DNA nanotubes of controlled diameters can be formed. The formation of the DNA tubes, and the control over the diameters of the generated nanotubes, are attributed to the thermodynamically favoured unidirectional growth of the sheets of the respective subunits, followed subjected to the folding of sheets by elastic-energy penalties that are compensated by favoured binding energies.

Autonomous Replication of Nucleic Acids by Polymerization/Nicking Enzyme/DNAzyme Cascades for the Amplified Detection of DNA and the Aptamer–Cocaine Complex

The progressive development of amplified DNA sensors and aptasensors using replication/nicking enzymes/DNAzyme machineries is described. The sensing platforms are based on the tailoring of a DNA template on which the recognition of the target DNA or the formation of the aptamer-substrate complex trigger on the autonomous isothermal replication/nicking processes and the displacement of a Mg(2+)-dependent DNAzyme that catalyzes the generation of a fluorophore-labeled nucleic acid acting as readout signal for the analyses. Three different DNA sensing configurations are described, where in the ultimate configuration the target sequence is incorporated into a nucleic acid blocker structure associated with the sensing template. The target-triggered isothermal autonomous replication/nicking process on the modified template results in the formation of the Mg(2+)-dependent DNAzyme tethered to a free strand consisting of the target sequence. This activates additional template units for the nucleic acid self-replication process, resulting in the ultrasensitive detection of the target DNA (detection limit 1 aM). Similarly, amplified aptamer-based sensing platforms for cocaine are developed along these concepts. The modification of the cocaine-detection template by the addition of a nucleic acid sequence that enables the autonomous secondary coupled activation of a polymerization/nicking machinery and DNAzyme generation path leads to an improved analysis of cocaine (detection limit 10 nM).

Nucleic acid driven DNA machineries synthesizing Mg2+-dependent DNAzymes: an interplay between DNA sensing and logic-gate operations.

Polymerase/nicking enzymes and nucleic-acid scaffolds are implemented as DNA machines for the development of amplified DNA-detection schemes, and for the design of logic gates. The analyte nucleic acid target acts, also, as input for the logic gates. In the presence of two DNA targets, acting as inputs, and appropriate DNA scaffolds, the polymerase-induced replication of the scaffolds, followed by the nicking of the replication products, are activated, leading to the autonomous synthesis of the Mg(2+)-dependent DNAzyme or the Mg(2+)-dependent DNAzyme subunits. These biocatalysts cleave a fluorophore/quencher-functionalized nucleic-acid substrate, thus providing fluorescence signals for the sensing events or outputs for the logic gates. The systems are used to develop OR, AND, and Controlled-AND gates, and the DNA-analyte targets represent two nucleic acid sequences of the smallpox viral genome.

DNAzyme-based 2:1 and 4:1 multiplexers and 1:2 demultiplexer

Scaffolding proteins play a central role in many regulatory cellular networks, where signalling proteins trigger different, and even orthogonal biological pathways. Such biological regulatory networks can be duplicated by multiplexer/demultiplexer logic operations. We present the use of libraries of Mg2+-dependent DNAzyme subunits as computational moduli for the construction of 2:1 and 4:1 multiplexers and a 1:2 demultiplexer. In the presence of the appropriate inputs, and the presence or absence of selector units, the guided assembly of the DNAzyme subunits to form active Mg2+-dependent DNAzyme proceeds. The formation of the active DNAzyme nanostructures is controlled by the energetics associated with the resulting duplexes between the inputs/selectors and the DNAzyme subunits. The library subunits are designed in such a way that, in the presence of the appropriate inputs/selectors, the inputs are knocked-down or triggered-on to yield the respective multiplexer/demultiplexer operations. Fluorescence is used as the readout for the outputs of the logic operations. The DNAzyme-based multiplexer/demultiplexer systems present biomolecular assemblies for data compression and decompression.