Functional patterning of DNA origami by parallel enzymatic modification

We demonstrate here a rapid and cost-effective technique for nanoscale patterning of functional molecules on the surface of a DNA origami. The pattern is created enzymatically by transferring a functionalized dideoxynucleotide to the 3'-end of an arbitrary selected set of synthetic DNA oligonucleotides positioned approximately 6 nm apart in a 70 × 100 nm(2) rectangular DNA origami. The modifications, which are performed in a single-tube reaction, provide an origami surface modified with a variety of functional groups including chemical handles, fluorescent dyes, or ligands for subsequent binding of proteins. Efficient labeling and patterning was demonstrated by gel electrophoresis shift assays, reverse-phase HPLC, mass spectrometry, atomic force microscopy (AFM) analysis, and fluorescence measurements. The results show a very high yield of oligonucleotide labeling and incorporation in the DNA origami. This method expands the toolbox for constructing several different modified DNA origami from the same set of staple strands.     

Rapid prototyping of arbitrary 2D and 3D wireframe DNA origami

Wireframe DNA origami assemblies can now be programmed automatically from the top-down using simple wireframe target geometries, or meshes, in 2D and 3D, using either rigid, six-helix bundle (6HB) or more compliant, two-helix bundle (DX) edges. While these assemblies have numerous applications in nanoscale materials fabrication due to their nanoscale spatial addressability and high degree of customization, no easy-to-use graphical user interface software yet exists to deploy these algorithmic approaches within a single, standalone interface. Further, top-down sequence design of 3D DX-based objects previously enabled by DAEDALUS was limited to discrete edge lengths and uniform vertex angles, limiting the scope of objects that can be designed. Here, we introduce the open-source software package ATHENA with a graphical user interface that automatically renders single-stranded DNA scaffold routing and staple strand sequences for any target wireframe DNA origami using DX or 6HB edges, including irregular, asymmetric DX-based polyhedra with variable edge lengths and vertices demonstrated experimentally, which significantly expands the set of possible 3D DNA-based assemblies that can be designed. ATHENA also enables external editing of sequences using caDNAno, demonstrated using asymmetric nanoscale positioning of gold nanoparticles, as well as providing atomic-level models for molecular dynamics, coarse-grained dynamics with oxDNA, and other computational chemistry simulation approaches.     

Reconfiguration of DNA nanostructures induced by enzymatic ligation treatment

Enzymatic ligation is a popular method in DNA nanotechnology for structural enforcement. When employed as stability switch for chosen components, ligation can be applied to induce DNA nanostructure reconfiguration. In this study, we investigate the reinforcement effect of ligation on addressable DNA nanostructures assembled entirely from short synthetic strands as the basis of structural reconfiguration. A careful calibration of ligation efficiency is performed on structures with programmable nicks. Systematic investigation using comparative agarose gel electrophoresis enables quantitative assessment of enhanced survivability with ligation treatment on a number of unique structures. The solid ligation performance sets up the foundation for the ligation-based structural reconfiguration. With the capability of switching base pairing status between permanent and transient (ON and OFF) by a simple round of enzymatic treatment, ligation induced reconfiguration can be engineered for DNA nanostructures accordingly.     

Fidelity of enzymatic ligation for DNA computing.

We describe a convenient assay for rapid qualitative evaluation of hybridization/ligation fidelity. The approach uses randomized probe strands of DNA and restriction enzyme digestion after amplification of reaction products by the polymerase chain reaction (PCR). We report ligation efficiencies and fidelities of two DNA ligases, T4 DNA ligase and Thermus aquaticus (Taq) DNA ligase, over a range of temperatures.     

Conformation of ring single-stranded DNA measured by DNA origami structures

Measuring the mechanical properties of single-stranded DNA (ssDNA) is a complex challenge that has been addressed lately by different methods. We measured the persistence length of ring ssDNA using a combination of a special DNA origami structure, a self-avoiding ring polymer simulation model, and nonparametric estimation statistics. The method overcomes the complexities set forth by previously used methods. We designed the DNA origami nano structures and measured the ring ssDNA polymer conformations using atomic force microscopy. We then calculated their radius of gyration, which was used as a fitting parameter for finding the persistence length. As there is no simple formulation for the radius of gyration distribution, we developed a simulation program consisting of a self-avoiding ring polymer to fit the persistence length to the experimental data. ssDNA naturally forms stem-loops, which should be taken into account in fitting a model to the experimental measurement. To overcome that hurdle, we found the possible loops using minimal energy considerations and used them in our fitting procedure of the persistence length. Due to the statistical nature of the loops formation, we calculated the persistence length for different percentages of loops that are formed. In the range of 25–75% loop formation, we found the persistence length to be 1.9–4.4 nm, and for 50% loop formation we get a persistence length of 2.83 ± 0.63 nm. This estimation narrows the previously known persistence length and provides tools for finding the conformations of ssDNA.     

Nanopores formed by DNA origami: A review

Nanopores have emerged over the past two decades to become an important technique in single molecule experimental physics and biomolecule sensing. Recently DNA nanotechnology, in particular DNA origami, has been used for the formation of nanopores in insulating materials. DNA origami is a very attractive technique for the formation of nanopores since it enables the construction of 3D shapes with precise control over geometry and surface functionality. DNA origami has been applied to nanopore research by forming hybrid architectures with solid state nanopores and by direct insertion into lipid bilayers. This review discusses recent experimental work in this area and provides an outlook for future avenues and challenges.     

Investigation of DNA structural changes by infrared spectroscopy

DNA-oriented samples of various origins were studied under different conditions of humidiity and sodium chloride content by means of infrared spectroscopy. (1) Oriented DNA (M. Lysodeikticus, E. coli, calf thymus and salmon sperm) films at 3–4% sodium chloride yield polarized spectra which show drastic changes at relative humidities (r.h.) between 94% and 0% indicative of conformational changes: B form → a form → disordered form The measurements of the infrared dichroism at frequencies of about 1230 cm^?1 and at about 1090 cm^?1 allow one to determine the orientation of the phosphate group, whereas the measurements at 1710 cm^?1 characterize the base orientation. At humidities higher than 90% r.h. (B form) the bisector of OPO forms an angle of 70° relative to the helix axis, whereas at lower humidities, between 75% and 50% r.h. (A form) a rotation to about 45° is observed. Simultaneously, the 0—0 line of phosphate group changes its orientation from 55° to 65° to the helix when B → A transition takes place. The results are in general agreement with that of X-ray diffraction and allow one to determine the orientation of the phosphate group with greater precision. (2) The B–A conformational change is not observed for satellite DNA, isolated from Cancer pagurus, of which the guanine + cytosine content is below 5%. As a function of decreasing humidities, one observes the transition: B form → disordered form A diagram of conformational changes of DNA's as a function of base composition and of r.h., suggests that B–A transition will occur for DNA of relatively higher G + C content, whereas for high (A + T) content, base sequence may be of importance. The B–A transition is prevented in DNA at a relatively high or very low sodium chloride content.     

Aggregation of DNA by analogs of spermidine; enzymatic and structural studies.

A homologous series of spermidine analogs, with defined abilities to replace the natural polyamine in supporting cell growth, was examined for its influence on the structure of supercoiled, aggregated DNA and on the ability of the DNA aggregates to act as substrates for various enzymes. The concentration of amine necessary to aggregate negatively supercoiled Col E1 DNA was progressively increased as the diaminobutane moiety of spermidine was extended beyond 5 methylene groups. 1H- and 31P-NMR spectroscopy suggested that less rigid DNA aggregates were formed by spermidine analogs than by spermidine itself. Spermidine and its analogs differentially modulated the activities of bacterial and mammalian type I topoisomerases and EcoRI restriction endonuclease on aggregated DNA in a manner reminiscent of the abilities of the amines to stimulate cell growth. When DNA was not aggregated, the influence of the various amines on these reactions was almost identical. These results are discussed in relation to the structures of the DNA aggregates in the presence of the various triamines.     

Oral D2O administration and enzymatic changes in rat testis.

Heavy water caused marked gradual decrease in the weight of the body as well as the testes throughout the treatment interval ranging from 1 to 6 weeks. Following D2O oral administration, an overall significant fall in the activity of acid phosphatase and glucose-6-phosphatase was registered. On the other hand, the activity of lactic and succinic dehydrogenases, alkaline phosphatase and adenosine triphosphatase increases following D2O treatment. These changes in the enzyme activity are suggestive of an altered metabolism of the testes in response to D2O administration. Our data corroborate the view that biological systems do discriminate between H2 and D2.     

Energy landscapes of rotary DNA origami devices determined by fluorescence particle tracking

Biomolecular nanomechanical devices are of great interest as tools for the processing and manipulation of molecules, thereby mimicking the function of nature’s enzymes. DNA nanotechnology provides the capability to build molecular analogs of mechanical machine elements such as joints and hinges via sequence-programmable self-assembly, which are otherwise known from traditional mechanical engineering. Relative to their size, these molecular machine elements typically do not reach the same relative precision and reproducibility that we know from their macroscopic counterparts; however, as they are scaled down to molecular sizes, physical effects typically not considered by mechanical engineers such as Brownian motion, intramolecular forces, and the molecular roughness of the devices begin to dominate their behavior. In order to investigate the effect of different design choices on the roughness of the mechanical energy landscapes of DNA nanodevices in greater detail, we here study an exemplary DNA origami-based structure, a modularly designed rotor-stator arrangement, which resembles a rotatable nanorobotic arm. Using fluorescence tracking microscopy, we follow the motion of individual rotors and record their corresponding energy landscapes. We then utilize the modular construction of the device to exchange its constituent parts individually and systematically test the effect of different design variants on the movement patterns. This allows us to identify the design parameters that most strongly affect the shape of the energy landscapes of the systems. Taking into account these insights, we are able to create devices with significantly flatter energy landscapes, which translates to mechanical nanodevices with improved performance and behaviors more closely resembling those of their macroscopic counterparts.     

DNA origami signposts for identifying proteins on cell membranes by electron cryotomography

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Evidence of structural changes of an enzymatic extract entrapped into alginate beads

In this work, we analyzed the structural changes of araujiain entrapped into alginate beads. Araujiain is an enzymatic preparation containing three known enzymatic fractions with each fraction individually presenting a similar catalytic performance. Fluorescence and infrared spectroscopy, thermal analysis and residual catalytic activity studies were carried out. A small red shift in the spectrum of araujiain was observed after the entrapment process. Changes in the polarity around the tryptophan (Trp) residues were associated with an enzyme conformational change. From the Fourier transform infrared spectroscopy (FTIR) analysis, it was demonstrated that interactions between the enzyme extract and Ca alginate caused different structural behavior in araujiain. According to the diffuse reflectance infrared Fourier transform spectroscopy (DRIFT) study, it was possible to conclude that a secondary structure with a high α-helical character was responsible for the highest activity of entrapped araujiain. Finally, from thermal analysis measurements, it was proved that entrapment of araujiain augments the thermal stability of both the enzyme extract and Ca alginate, indicating a possible interaction between enzyme extract and its support.     

Template-independent ligation of single-stranded DNA by T4 DNA ligase

T4 DNA ligase is one of the workhorses of molecular biology and used in various biotechnological applications. Here we report that this ligase, unlike Escherichia coli DNA ligase, Taq DNA ligase and Ampligase, is able to join the ends of single-stranded DNA in the absence of any duplex DNA structure at the ligation site. Such nontemplated ligation of DNA oligomers catalyzed by T4 DNA ligase occurs with a very low yield, as assessed by quantitative competitive PCR, between 10(-6) and 10(-4) at oligonucleotide concentrations in the range 0.1-10 nm, and thus is insignificant in many molecular biological applications of T4 DNA ligase. However, this side reaction may be of paramount importance for diagnostic detection methods that rely on template-dependent or target-dependent DNA probe ligation in combination with amplification techniques, such as PCR or rolling-circle amplification, because it can lead to nonspecific background signals or false positives. Comparison of ligation yields obtained with substrates differing in their strandedness at the terminal segments involved in ligation shows that an acceptor duplex DNA segment bearing a 3'-hydroxy end, but lacking a 5'-phosphate end, is sufficient to play a role as a cofactor in blunt-end ligation.     

Stabilization of microtubules by Combretastatin D derivatives

The effect of five derivatives of Combretastatin D on tubulin polymerization was investigated. All of them were found to stabilize microtubules to various degrees. The derivatives bearing polar substituents were found to be the most active.