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Genomic integrity, when compromised by accrued DNA lesions, is maintained through efficient repair via homologous recombination. For this process the ubiquitous recombinase A (RecA), and its homologues such as the human Rad51, are of central importance, able to align and exchange homologous sequences within single-stranded and double-stranded DNA in order to swap out defective regions. Here, we directly observe the widely debated mechanism of RecA homology searching at a single-molecule level using high-speed atomic force microscopy (HS-AFM) in combination with tailored DNA origami frames to present the reaction targets in a way suitable for AFM-imaging. We show that RecA nucleoprotein filaments move along DNA substrates via short-distance facilitated diffusions, or slides, interspersed with longer-distance random moves, or hops. Importantly, from the specific interaction geometry, we find that the double-stranded substrate DNA resides in the secondary DNA binding-site within the RecA nucleoprotein filament helical groove during the homology search. This work demonstrates that tailored DNA origami, in conjunction with HS-AFM, can be employed to reveal directly conformational and geometrical information on dynamic protein-DNA interactions which was previously inaccessible at an individual single-molecule level.

Under a layer of 0.1 M HCl in isopropanol, soft ultraviolet (UV) (365 nm) photolysis of the thiol-on-gold self-assembled monolayer (SAM) derived from the lipoic acid ester of α-hydroxy-1-acetylpyrene results in the expected removal of the acetylpyrene protecting group. When photolyzing through a mask, this can be used to produce a patterned surface and, at a controlled electrochemical potential, it is then possible to selectively and reversibly electrodeposit copper on the photolyzed regions. Rather surprisingly, under these photolysis conditions, there is not only the expected photodeprotection of the ester but also partial removal of the lipoic acid layer which has been formed. In further studies, it is shown that this type of acid-catalyzed photoremoval of SAM layers by soft UV is a rather general phenomenon and results in the partial removal of the thiol-on-gold SAM layers derived from other ω-thiolated carboxylic acids. However, this phenomenon is chain-length dependent. Under conditions in which there is a ∼60% reduction in the thickness of the SAM derived from dithiobutyric acid, the SAM derived from mercaptoundecanoic acid is almost unaffected. The process by which the shorter-chain SAM layers are partially removed is not fully understood because these compounds do not absorb significantly in the 365 nm region of the spectrum! Significantly, this study shows that acid catalysis photolysis of thiol-on-gold SAMs needs to be used with caution.

Protein-mediated self-assembly is arguably one of the most promising routes for building complex molecular nanostructures. Here, we report a molecular self-assembly technique that allows programmable, site-specific patterning of double-stranded DNA scaffolds, at a single-base resolution, by 3-nm-long RecA-based nucleoprotein filaments. RecA proteins bind to single-stranded DNA to form nucleoprotein filaments. These can self-assemble onto a double-stranded DNA scaffold at a region homologous to the nucleoprotein's single-stranded DNA sequence. We demonstrate that nucleoprotein filaments can be formed from single-stranded DNA molecules ranging in length from 60 nucleotides down to just 6 nucleotides, and these can be assembled site-specifically onto a model DNA scaffold both at the end of the scaffold and away from the end. In both cases, successful site-specific self-assembly is demonstrated even for the smallest nucleoprotein filaments, which are just 3 nm long, comprise only two monomers of RecA, and cover less than one helical turn of the double-stranded DNA scaffold. Finally, we demonstrate that the RecA-mediated assembly process is highly site-specific and that the filaments indeed bind only to the homologous region of the DNA scaffold, leaving the neighboring scaffold exposed.

Self-assembled monolayers (SAMs) can be formed at the interface between solids and fluids, and are often used to modify the surface properties of the solid. One of the most widely employed SAM systems is exploiting thiol-gold chemistry, which, together with alkane-chain-based molecules, provides a reliable way of SAM formation to modify the surface properties of electrodes. Oligo ethylene-glycol (OEG) terminated alkanethiol monolayers have shown excellent antifouling properties and have been used extensively for the coating of biosensor electrodes to minimize nonspecific binding. Here, we report the investigation of the dielectric properties of COOH-capped OEG monolayers and demonstrate a strategy to improve the dielectric properties significantly by mixing the OEG SAM with small concentrations of 11-mercaptoundecanol (MUD). The monolayer properties and composition were characterized by means of impedance spectroscopy, water contact angle, ellipsometry and X-ray photoelectron spectroscopy. An equivalent circuit model is proposed to interpret the EIS data and to determine the conductivity of the monolayer. We find that for increasing MUD concentrations up to about 5% the resistivity of the SAM steadily increases, which together with a considerable decrease of the phase of the impedance, demonstrates significantly improved dielectric properties of the monolayer. Such monolayers will find widespread use in applications which depend critically on good dielectric properties such as capacitive biosensor.

The coiled coil is a widespread protein motif responsible for directing the assembly of a wide range of protein complexes. To date, research has focused largely on the solution phase assembly of coiled-coil complexes. Here, we describe an investigation into coiled-coil heterodimer assembly where one of the peptides is immobilized directly onto a gold electrode. Immobilization is achieved by the introduction of a unique cysteine residue at the C terminus, allowing for covalent and orientated attachment to a thiol-reactive surface, here the gold electrode. We show an electrochemical impedance of the resulting self-assembled polypeptide monolayer around |Z| = 4 × 10(4) Ω cm(2) at 100 mHz with a minimum phase angle of -84°, consistent with the formation of a densely packed, insulating layer. The thickness of the peptide monolayer, as measured using ellipsometry, is around 3 nm, close to that expected for a self-assembled monolayer assembled from helical peptides. Crucially, we find that the efficiency of dimerization between a peptide in solution and its coiled-coil partner peptide immobilized on a surface is strongly dependent upon the density of the immobilized peptide layer, with dimer assembly being strongly suppressed by high-density peptide monolayers. We thus develop an approach for controlling the density of the immobilized peptide by diluting the monolayer with a thiolated, random-coil peptide to modulate dimerization efficiency and demonstrate electrochemical detection of highly specific, coiled-coil heterodimer on-surface assembly.

We discuss the assembly, structure, and stability of multilayer molecular films formed from multiple mercaptoalkanoic acid monolayers ligated via carboxyl and thiol interactions with divalent copper ions. Using dual-polarization interferometry to study the assembly of multilayer films in real time, we observe a clear linear relationship between the number of layers within a film and the overall average film thickness. Unexpectedly, however, we find a restructuring of the lower monolayer upon association of the Cu(2+) ions to form the Cu carboxylate surface. In particular, the thickness of the lower monolayer was found to decrease significantly, accompanied by an increase in the film density. The conformation of the monolayer subsequently recovered to that observed originally following the reduction of the Cu ion to Cu(+) upon chemisorption of the adlayer. Comparable restructuring was also observed in molecular dynamics simulations of a bilayer film assembled on a gold surface. Our combined experimental and theoretical study suggests that the observed restructuring is a result of charge-charge interactions between adjacent Cu ions that exist in the +2 oxidation state in the copper carboxylate surface and in the +1 oxidation state following chemisorption of the adlayer.

The structural stability of alkenthiolate monolayers assembled on gold surfaces is a result of the well-defined organization of the individual molecules within the film. The formation of three-dimensional films assembled by stacking multiple molecular monolayers is substantially more challenging because the correct organization of the molecular components is required not only within the individual monolayers but also between the monolayers of the film. In this paper we examine the structure of multilayer films based on mercaptoalkanoic acid monolayers in which ligation between adjacent monolayers is achieved using the interaction of carboxylic acid and thiol groups with a divalent Cu ion. Using contact angle analysis and atomic force microscopy, we show that the use of Cu(2+) has profound implications on the properties and structure of the multilayer film. In particular, the divalent ions effectively prohibit the complete assembly of the next monolayer. For multilayer SAMs assembled from short alkane chains with six methylene groups, we find that molecules in the incomplete adlayer organize themselves randomly over the underlying monolayer. However, as the number of methylene groups increases (11 and 16 methylene groups), the upper layer tends to fracture into discrete islands which cover around 50% of the surface. The height of these islands is found to be equal to that expected for a complete, well-ordered monolayer assembled from the equivalent mercaptoalkanoic acid molecules. This relationship between chain length and island growth results from the migration of molecules into ordered aggregates driven by the reduction of free energy associated with maximizing intermolecular interactions.

We report on the development of novel cyano-biphenyl-based thiolate self-assembled monolayers designed to promote homeotropic alignment of calamitic liquid crystals. The molecules developed contain an ortho-nitrobenzyl protected carboxylic acid group that on irradiation by soft UV (365 nm) is cleaved to yield carboxylic acid groups exposed at the surface that promote planar alignment. Using a combination of wetting, X-ray photoelectron spectroscopy, Fourier transform-infrared reflection absorption spectroscopy, and ellipsometry we show that high photolysis yields (>90%) can be achieved and that the patterned SAMs are suitable for the controlled alignment of calamitic liquid crystals. This study further shows that such photo-patterned SAMs can be used to control the formation of focal conic domains (FCDs) in the smectic-A phase in terms of positioning and size confinement on surfaces.

We demonstrate the use of surface-immobilized, oriented peptide aptamers for the detection of specific target proteins from complex biological solutions. These peptide aptamers are target-specific peptides expressed within a protein scaffold engineered from the human protease inhibitor stefin A. The scaffold provides stability to the inserted peptides and increases their binding affinity owing to the resulting three-dimensional constraints. A unique cysteine residue was introduced into the protein scaffold to allow orientation-specific surface immobilization of the peptide aptamer and to ensure exposure of the binding site to the target solution. Using dual-polarization interferometry, we demonstrate a strong relationship between binding affinity and aptamer orientation and determine the affinity constant KD for the interaction between an oriented peptide aptamer ST(cys+)_(pep9) and the target protein CDK2. Further, we demonstrate the high selectivity of the peptide aptamer STM_(pep9) by exposing surface-immobilized ST(cys+)_(pep9) to a complex biological solution containing small concentrations of the target protein CDK2.

The inherent self-recognition properties of DNA have led to its use as a scaffold for various nanotechnology self-assembly applications, with macromolecular complexes, metallic and semiconducting nanoparticles, proteins, inter alia, being assembled onto a designed DNA scaffold. Such structures may typically comprise a number of DNA molecules organized into macromolecules. Many studies have used synthetic methods to produce the constituent DNA molecules, but this typically constrains the molecules to be no longer than around 100 base pairs (30 nm). However, applications that require larger self-assembling DNA complexes, several tens of nanometers or more, need to be generated by other techniques. Here, we present a generic technique to generate large linear, branched, and/or circular DNA macromolecular complexes. The effectiveness of this technique is demonstrated here by the use of Lambda Bacteriophage DNA as a template to generate single- and double-branched DNA structures approximately 120 nm in size.