Ofer I. Wilner

A polycatenated DNA scaffold for the one-step assembly of hierarchical nanostructures

A unique DNA scaffold was prepared for the one-step self-assembly of hierarchical nanostructures onto which multiple proteins or nanoparticles are positioned on a single template with precise relative spatial orientation. The architecture is a topologically complex ladder-shaped polycatenane in which the “rungs” of the ladder are used to bring together the individual rings of the mechanically interlocked structure, and the “rails” are available for hierarchical assembly, whose effectiveness has been demonstrated with proteins, complementary DNA, and gold nanoparticles. The ability of this template to form from linear monomers and simultaneously bind two proteins was demonstrated by chemical force microscopy, transmission electron microscopy, and confocal fluorescence microscopy. Finally, fluorescence resonance energy transfer between adjacent fluorophores confirmed the programmed spatial arrangement between two different nanomaterials. DNA templates that bring together multiple nanostructures with precise spatial control have applications in catalysis, biosensing, and nanomaterials design.

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.

Probing Kinase Activities by Electrochemistry, Contact‐Angle Measurements, and Molecular‐Force Interactions

Three different methods to investigate the activity of a protein kinase (casein kinase, CK2) are described. The phosphorylation of the sequence-specific peptide (1) by CK2 was monitored by electrochemical impedance spectroscopy (EIS). Phosphorylation of the peptide monolayer assembled on a Au electrode yields a negatively charged surface that electrostatically repels the negatively charged redox label [Fe(CN)6]3-/4-, thus increasing the interfacial electron-transfer resistance. The phosphorylation process by CK2 is further amplified by the association of the anti-phosphorylated peptide antibody to the monolayer. Binding of the antibody insulates the electrode surface, thus increasing the interfacial electron-transfer resistance in the presence of the redox label. This method enabled the quantitative analysis of the concentration of CK2 with a detection limit of ten units. The second method employed involved contact-angle measurements. Although the peptide 1-functionalized electrode revealed a contact angle of 67.5 degrees , phosphorylation of the peptide yielded a surface with enhanced hydrophilicity, 36.8 degrees. The biocatalyzed cleavage of the phosphate units with alkaline phosphatase regenerates the hydrophobic peptide monolayer, contact angle 55.3 degrees . The third method to characterize the CK2 system involved chemical force measurements between the phosphorylated peptide monolayer associated with the Au surface and a Au tip functionalized with the anti-phosphorylated peptide antibody. Although no significant rupture forces existed between the modified tip and the 1-functionalized surface (6+/-2 pN), significant rupture forces (multiples of 120+/-20 pN) were observed between the phosphorylated monolayer-modified surface and the antibody-functionalized tip. This rupture force is attributed to the dissociation of a simple binding event between the phosphorylated peptide and the fluorescent antibody (Fab) binding region.

APOBEC3G enhances lymphoma cell radioresistance by promoting cytidine deaminase-dependent DNA repair

APOBEC3 proteins catalyze deamination of cytidines in single-stranded DNA (ssDNA), providing innate protection against retroviral replication by inducing deleterious dC > dU hypermutation of replication intermediates. APOBEC3G expression is induced in mitogen-activated lymphocytes; however, no physiologic role related to lymphoid cell proliferation has yet to be determined. Moreover, whether APOBEC3G cytidine deaminase activity transcends to processing cellular genomic DNA is unknown. Here we show that lymphoma cells expressing high APOBEC3G levels display efficient repair of genomic DNA double-strand breaks (DSBs) induced by ionizing radiation and enhanced survival of irradiated cells. APOBEC3G transiently accumulated in the nucleus in response to ionizing radiation and was recruited to DSB repair foci. Consistent with a direct role in DSB repair, inhibition of APOBEC3G expression or deaminase activity resulted in deficient DSB repair, whereas reconstitution of APOBEC3G expression in leukemia cells enhanced DSB repair. APOBEC3G activity involved processing of DNA flanking a DSB in an integrated reporter cassette. Atomic force microscopy indicated that APOBEC3G multimers associate with ssDNA termini, triggering multimer disassembly to multiple catalytic units. These results identify APOBEC3G as a prosurvival factor in lymphoma cells, marking APOBEC3G as a potential target for sensitizing lymphoma to radiation therapy.

Control of Bioelectrocatalytic Transformations on DNA Scaffolds

The spatial organization of biomolecules on a DNA scaffold linked to an electrode leads to programmed biocatalytic transformations. This is exemplified by the electrical contacting of glucose oxidase (GOx) linked to the DNA scaffold with the electrode. A nucleic acid functionalized with a ferrocene relay unit was hybridized with the DNA scaffold at a position adjacent to the electrode, and GOx functionalized with nucleic acid units complementary to the specific domain of the DNA template was hybridized with the DNA scaffold in a position remote from the electrode. Under these conditions, ferrocene-mediated oxidation of the redox center of GOx occurred, and the effective bioelectrocatalytic oxidation of glucose was activated. Exchange of the position of GOx and the electron-mediator groups prohibited the bioelectrocatalytic oxidation of glucose. In another system, a nucleic acid-functionalized microperoxidase-11 (MP-11) and the nucleic acid-modified GOx were hybridized with the adjacent and remote sites, respectively, on the DNA scaffold associated with the electrode. In this configuration, effective MP-11-catalyzed reduction of H(2)O(2) generated by the GOx-catalyzed oxidation of glucose occurred, and the resulting bioelectrocatalytic cathodic currents were controlled by the concentration of glucose. Exchanging the positions of MP-11 and GOx on the DNA scaffold eliminated the MP-11-electrocatalyzed reduction of H(2)O(2).

Covalently Linked DNA Nanotubes

The present study introduces an approach to prepare covalently linked DNA nanotubes. A circular DNA that includes at its opposite poles thiol and amine functionalities acts as the building block for the construction of the DNA nanotubes. The circular DNA is cross-linked with a bis-amide-modified nucleic acid to yield DNA nanowires, and these are subsequently cross-linked by a bis-thiolated nucleic acid to yield the DNA nanotubes. Alternatively, a circular DNA that includes four amine functionalities on its poles is cross-linked in one-step by the bis-thiolated nucleic acid to yield the nanotubes. The resulting nanostructures are stable and nonseparable upon heating.

Photoelectrochemical cells based on bis-aniline-crosslinked CdS nanoparticle–carbon nanotube matrices associated with electrodes

The electrochemical preparation of a bis-aniline-crosslinked CdS nanoparticle–carbon nanotube matrix on electrode surfaces is described. The optimal electrode was prepared by the electropolymerization of thioaniline-functionalized CdS nanoparticles (NPs) and aniline-tethered carbon nanotubes (CNTs), using 80 electropolymerization cycles at a CdS NPs:CNTs (w/w) ratio of 5.5. The photocurrent generated by the electrode, in the presence of triethanolamine as electron-donor, reveals a quantum yield of ϕ = 2.4%, ca. seven-fold higher than for a monolayer of CdS NPs crosslinked to the electrode by bis-aniline bridges. The enhanced photocurrents in the CdS NP–CNT composite were attributed to the trapping of the photogenerated conduction-band electrons in the semiconductor NPs by the CNTs, and their effective transport to the electrode, a process that facilitated charge separation. The bias potential applied to the electrodes affected the resulting photocurrents, and enhanced photocurrents were observed when the bis-aniline bridges existed in their oxidized quinoid state. This was attributed to the improved trapping of the conduction-band electrons by the electron-acceptor (relay) bridging units. The supramolecular association of N,N′-dimethyl-4,4′-bipyridinium, MV2+, to the π-donor bis-aniline bridging units resulted in a photocurrent quantum yield of ϕ = 6.1%. The enhanced quantum yield was attributed to the effective trapping of the conduction-band electrons by the π-acceptor MV2+ relay units associated with the π-donor bridging elements, and the efficient transport of the electrons to the electrode by the conductive CNT matrix. These processes facilitated and improved charge separation and provided a competitive path to degradative electron–hole recombination in the semiconductor particles.