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DNA origami synthesis is a well-established technique with wide-ranging applications. In most cases, the synthesized origami must be purified to remove excess materials such as DNA oligos and other functional molecules. While several purification techniques are routinely used, all have limitations, and cannot be integrated with robotic systems. Here the use of solid-phase reversible immobilization (SPRI) beads as a scalable, high-throughput, and automatable method to purify DNA origami is demonstrated. Not only can this method remove unreacted oligos and biomolecules with yields comparable to existing methods while maintaining the high structural integrity of the origami, but it can also be integrated into an automated workflow to purify simultaneously large numbers and quantities of samples. It is envisioned that the SPRI beads purification method will improve the scalability of DNA nanostructures synthesis both for research and commercial applications., (© 2023 The Authors. Small published by Wiley‐VCH GmbH.)

The ability to detect low concentrations of biomarkers in patient samples is one of the cornerstones of modern healthcare. In general, biosensing approaches are based on measuring signals resulting from the interaction of a large ensemble of molecules with the sensor. Here, we report a biosensor platform using DNA origami featuring a central cavity with a target-specific DNA aptamer coupled with a nanopore read-out to enable individual biomarker detection. We show that the modulation of the ion current through the nanopore upon the DNA origami translocation strongly depends on the presence of the biomarker in the cavity. We exploit this to generate a biosensing platform with a limit of detection of 3 nM and capable of the detection of human C-reactive protein (CRP) in clinically relevant fluids. Future development of this approach may enable multiplexed biomarker detection by using ribbons of DNA origami with integrated barcoding.

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.

The mechanism by which pre-synaptic RecA nucleoprotein filaments efficiently locate sequence homology across genomic DNA remains unclear. Here, using atomic force microscopy, we directly investigate the intermediates of the RecA-mediated homologous recombination process and find it to be highly cooperative, involving multiple phases. Initially, the process is dominated by a rapid 'association' phase, where multiple filaments interact on the same dsDNA simultaneously. This cooperative nature is reconciled by the observation of localized dense clusters of pre-synaptic filaments interacting with the observed dsDNA molecules. This confinement of reactive species within the vicinity of the dsDNA, is likely to play an important role in ensuring that a high interaction rate between the nucleoprotein filaments and the dsDNA can be achieved. This is followed by a slower 'resolution' phase, where the synaptic joints either locate sequence homology and progress to a post-synaptic joint, or dissociate from the dsDNA. Surprisingly, the number of simultaneous synaptic joints decreases rapidly after saturation of the dsDNA population, suggesting a reduction in interaction activity of the RecA filaments. We find that the time-scale of this decay is in line with the time-scale of the dispersion of the RecA filament clusters, further emphasising the important role this cooperative phenomena may play in the RecA-facilitated homology search., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The use of DNA as a structural material for nanometre-scale construction has grown extensively over the last decades. The development of more advanced DNA-based materials would benefit from a modular approach enabling the direct assembly of additional elements onto nanostructures after fabrication. RecA-based nucleoprotein filaments encapsulating short ssDNA have been demonstrated as a tool for highly efficient and fully programmable post-hoc patterning of duplex DNA scaffold. However, the underlying assembly process is not fully understood, in particular when patterning complex DNA topologies. Here, we report the effect of basepair-mismatched regions and single-strand nicks in the double-stranded DNA scaffold on the yield of RecA-based assembly. Significant increases in assembly yield are observed upon the introduction of unpaired basepairs directly adjacent to the assembly region. However, when the unpaired regions were introduced further from the assembly site the assembly yield initially decreased as the length of the unpaired region was increased. These results suggest that an unpaired region acts as a kinetic trap for RecA-based nucleoprotein filaments, impeding the assembly mechanism. Conversely, when the unpaired region is located directly adjacent to the assembly site, it leads to an increase in efficiency of RecA patterning owing to increased breathing of the assembly site.

We present a method for the specific, spatially targeted attachment of DNA molecules to lithographically patterned gold surfaces-demonstrated by bridging DNA strands across nanogap electrode structures. An alkanethiol self-assembled monolayer was employed as a molecular resist, which could be selectively removed via electrochemical desorption, allowing the binding of thiolated DNA anchoring oligonucleotides to each electrode. After introducing a bridging DNA molecule with single-stranded ends complementary to the electrode-tethered anchoring oligonucleotides, the positioning of the DNA molecule across the electrode gap, driven by self-assembly, occurred autonomously. This demonstrates control of molecule positioning with resolution limited only by the underlying patterned structure, does not require any alignment, is carried out entirely under biologically compatible conditions, and is scalable.

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.

Molecular self-assembly inherent to many biological molecules, in conjunction with suitable molecular scaffolds to facilitate programmable positioning of nanoscale objects, offers a promising approach for the integration of functional nanoscale complexes into macroscopic host devices. Here, we report the use of the protein RecA as a means of highly efficient programmable patterning of double-stranded (ds)DNA molecules with molecular-scale precision at specific locations along the DNA strand. RecA proteins form nucleoprotein filaments with single-stranded (ss)DNA molecules, which are chosen to be of sequence homologous to the desired binding region on the dsDNA scaffold. We show that the patterning yield can be in excess of 85% and we demonstrate that concurrent patterning of multiple locations on the same dsDNA scaffold can be achieved with separation between the assembled nucleoprotein filaments of less than 4 nm. This is an important prerequisite for this programmable and flexible DNA scaffold patterning technique to be employed in molecular- and nanoscale assembly applications.

Molecular lock-and-key functionality inherent to a large number of biological molecules is arguably one of the most promising potential routes for building complex nanostructures and eventually nano-electronic devices by self-assembly. Here, we report the use of the DNA-binding protein RecA as a tool for patterning DNA scaffolds site-specifically and in a programmable way at the nanometre-scale. This is demonstrated by patterning a 3 kilo-base-pair double-stranded DNA molecule at ten different locations with nanometre-sized RecA-based nucleoprotein filaments. This protein-based molecular patterning technique, which is site-specific, programmable, and scalable, offers a potential basis for molecular lithography applications, and opens previously inaccessible routes towards the fabrication of complex functional nano-electronic devices by self-assembly.

Core-shell magnetic nanoparticles have received significant attention recently and are actively investigated owing to their large potential for a variety of applications. Here, the synthesis and characterization of bimetallic nanoparticles containing a magnetic core and a gold shell are discussed. The gold shell facilitates, for example, the conjugation of thiolated biological molecules to the surface of the nanoparticles. The composite nanoparticles were produced by the reduction of a gold salt on the surface of pre-formed cobalt or magnetite nanoparticles. The synthesized nanoparticles were characterized using ultraviolet-visible absorption spectroscopy, transmission electron microscopy, energy dispersion X-ray spectroscopy, X-ray diffraction and super-conducting quantum interference device magnetometry. The spectrographic data revealed the simultaneous presence of cobalt and gold in 5.6±0.8 nm alloy nanoparticles, and demonstrated the presence of distinct magnetite and gold phases in 9.2±1.3 nm core-shell magnetic nanoparticles. The cobalt-gold nanoparticles were of similar size to the cobalt seed, while the magnetite-gold nanoparticles were significantly larger than the magnetic seeds, indicating that different processes are responsible for the addition of the gold shell. The effect on the magnetic properties by adding a layer of gold to the cobalt and magnetite nanoparticles was studied. The functionalization of the magnetic nanoparticles is demonstrated through the conjugation of thiolated DNA to the gold shell.

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.

The authors investigate the elongation and orientation of different-sized deoxyribose nucleic acid (DNA) molecules, tethered onto gold electrodes via a terminal thiol, under the influence of high frequency ac electric fields. The DNA molecules are elongated from a random coil into an extended conformation and orientated along the electric field lines as a result of the forces acting on the molecules during the application of the ac electric fields. Elongation was observed in the frequency range 100 kHz–1 MHz, with field strengths of 0.06–1.0 MV/m. Maximum elongation for all DNA fragments tested, irrespective of size, was found for frequencies between 200 and 300 kHz. The torque acting on the induced dipole in the DNA molecules, complemented by a directional bias force, opposite in direction to the dielectrophoretic force, provides the main contribution to the elongation process. The length of elongation is limited to either half the distance between opposing electrodes or to the contour length of the DNA, whichever is shorter. Further, the authors show that the normalized length of the elongated DNA molecules is independent of the contour length of the DNA. [ABSTRACT FROM AUTHOR]

Dielectrophoretic manipulation enables the positioning and orientation of DNA molecules for nanometer-scale applications. However, the dependence of the dielectrophoretic force and torque on the electric field magnitude and frequency has to be well characterised to realise fully the potential of this technique. DNA in solution is attracted to the strongest electric field gradient (i.e. the electrode edge)as a result of the dielectrophoretic force, while the dielectrophoretic torque aligns the DNA with its longest axis parallel to the electric field. In this work, the authors attached λ-DNA fragments (48 and 25 kilobases)to an array of gold microelectrodes via a terminal thiol bond and characterised the orientation and elongation as a function of electric field magnitude (0.1-0.8MV/m)and frequency (0.08-1.1MHz). Maximum elongation was observed between 200 and 500 kHz for the attached DNA. Dielectrophoresis is limited by thermal randomisation at electric fields below 0.1MV/m and by electrothermal effects above 0.7MV/m. The authors conclude that dielectrophoresis can be used to manipulate surface-immobilised DNA reproducibly. [ABSTRACT FROM AUTHOR]