Assembly of DNA Nanostructures with Branched Tris‐DNA

Branched tris‐DNA, in which two oligonucleotides of the same sequence and one other oligonucleotide of a different sequence are connected with a rigid central linker, was prepared chemically by using a DNA synthesizer. Two branched tris‐DNA molecules with complementary DNA sequences form dimer and tetramer as well as linear and spherical oligomer complexes. The complex formation was studied by UV/thermal denaturation, enzyme digestion, gel electrophoresis, and AFM imaging.     

Rational Design of pH‐Responsive DNA Motifs with General Sequence Compatibility

Reconfigurable molecular events are key to molecular machines. In response to external cues, molecular machines rearrange/change their structures to perform certain functions. Such machines exist in nature, for example cell surface receptors, and have been artificially engineered. To be able to build sophisticated and efficient molecular machines for an increasing range of applications, constant efforts have been devoted to developing new mechanisms of controllable structural reconfiguration. Herein, we report a general design principle for pH‐responsive DNA motifs for general DNA sequences (not limited to triplex or i‐motif forming sequences). We have thoroughly characterized such DNA motifs by polyacrylamide gel electrophoresis (PAGE) and fluorescence spectroscopy and demonstrated their applications in dynamic DNA nanotechnology. We expect that it will greatly facilitate the development of DNA nanomachines, biosensing/bioimaging, drug delivery, etc.     

Sequence‐Specific DNA Alkylation by Tandem Py–Im Polyamide Conjugates

Tandem N‐methylpyrrole? N‐methylimidazole (Py? Im) polyamides with good sequence‐specific DNA‐alkylating activities have been designed and synthesized. Three alkylating tandem Py? Im polyamides with different linkers, which each contained the same moiety for the recognition of a 10 bp DNA sequence, were evaluated for their reactivity and selectivity by DNA alkylation, using high‐resolution denaturing gel electrophoresis. All three conjugates displayed high reactivities for the target sequence. In particular, polyamide 1 , which contained a β‐alanine linker, displayed the most‐selective sequence‐specific alkylation towards the target 10 bp DNA sequence. The tandem Py? Im polyamide conjugates displayed greater sequence‐specific DNA alkylation than conventional hairpin Py? Im polyamide conjugates ( 4 and 5 ). For further research, the design of tandem Py? Im polyamide conjugates could play an important role in targeting specific gene sequences.     

Sequence‐Dependent dsDNA‐Templated Formation of Fluorescent Copper Nanoparticles

There are only a few systematic rules about how to selectively control the formation of DNA‐templated metal nanoparticles (NPs) by varying sequence combinations of double‐stranded DNA (dsDNA), although many attempts have been made. Herein, we develop a facile method for sequence‐dependent formation of fluorescent CuNPs by using dsDNA as templates. Compared with random sequences, AT sequences are better templates for highly fluorescent CuNPs. Other specific sequences, for example, GC sequences, do not induce the formation of CuNPs. These results shed light on directed DNA metallization in a sequence‐specific manner. Significantly, both the fluorescence intensity and the fluorescence lifetime of CuNPs can be tuned by the length or the sequence of dsDNA. In order to demonstrate the promising practicality of our findings, a sensitive and label‐free fluorescence nuclease assay is proposed.     

Heat‐Flow‐Driven Oligonucleotide Gelation Separates Single‐Base Differences

DNA phase transitions are often induced by the addition of condensation agents or by dry concentration. Herein, we show that the non‐equilibrium setting of a moderate heat flow across a water‐filled chamber separates and gelates DNA strands with single‐base resolution. A dilute mix of DNA with two slightly different gel‐forming sequences separates into sequence‐pure hydrogels under constant physiological solvent conditions. A single base change in a 36 mer DNA inhibits gelation. Only sequences with the ability to form longer strands are concentrated, further elongated, and finally gelated by length‐dependent thermal trapping. No condensation agents, such as multivalent ions, were added. Equilibrium aggregates from dry concentration did not show any sequence separation. RNA is expected to behave identically owing to its equal thermophoretic properties. The highly sequence‐specific phase transition points towards new possibilities for non‐equilibrium origins of life.     

Electrophoretic mobility of duplex DNA cross‐linked by mechlorethamine at a cytosine–cytosine mismatch pair

The common nitrogen mustard, mechlorethamine, can form a covalent cross‐link between the two bases of a cytosine–cytosine mismatch pair within a DNA duplex. The cross‐linked species can be readily separated from DNA monoadducts and unreacted strands using denaturing polyacrylamide gel electrophoresis. Here, using DNA 19 mer duplexes that are mechlorethamine cross‐linked at a C₄–C₃₅, C₇–C₃₂, C₁₀–C₂₉, or C₁₃–C₂₆ mismatch pair, we show that the denaturing polyacrylamide gel electrophoresis mobility of the cross‐linked species is particularly sensitive to the proximity of the C–C cross‐link to the duplex end. Species that are cross‐linked at a C₄–C₃₅ mismatch have greater mobilities than those cross‐linked at C₇–C₃₂ or C₁₃–C₂₆, and the species with a central C₁₀–C₂₉ cross‐link have the lowest mobility. The mobility is also dependent on the proximity of the cross‐link to a 5′‐³²P‐phosphate or a 5′‐fluorescein label. We interpret these results in terms of the conformational properties of the cross‐linked species in the denaturing gel. The results are consistent with the retention of partial duplex character at the end proximal to the cross‐link, with an influence on the mobility of the GC/AT ratio proximal to the cross‐link and at the duplex end, and a small but discernible effect of the label.     

Effect of topological asymmetry on the electrophoretic mobility of branched DNA structures with and without single-base mismatches

The electrophoretic mobility of three-arm star DNA structures with varying degrees of branch length asymmetry has been investigated in polyacrylamide (PAA) hydrogels. We report the effect of single-base mismatches, adjacent to the branch point, on the mobility of branched DNA with three different arm lengths. Branched DNA structures were formed using wild-type and mutated fragments of the p53 tumor suppressor gene, which is believed to play an important role in cancer development. Branching was directed at the site of several previously characterized mutations in exon 7 of p53. At a given gel concentration, the mobility of branched DNA with fully complementary base pairing is found to increase as the degree of branch length asymmetry is increased. Ferguson analysis of the gel electrophoresis data leads to a retardation coefficient that is strongly dependent on topology. This finding can be explained in terms of a minimum molecular cross-section for each molecule. Specifically, we show that structures with the smallest molecular cross-section can access more pores in the gel, which leads to higher mobility. Our results can also be understood by considering the rotational diffusivity of branched DNA. Asymmetric DNA stars with higher calculated rotational diffusivities also have higher mobilities. When a mutated base is present in junctions with low degrees of branch length asymmetry, adjacent to the branch point, the mobility increases in comparison to the fully complementary molecules. The reason for this increased mobility is unclear, here, we propose that the mismatched base introduces additional flexibility to the arm containing the mutation leading to higher conformational freedom and enhanced mobility in gels. When a mismatched base is present in junctions with high degrees of branch length asymmetry, the opposite result is obtained. Here, the mutated species has a lower mobility. This result is argued to arise from incomplete hybridization and/or frayed ends. Finally, we have shown that by using two of the branch point oligonucleotides as probe molecules, mutations known to occur at specific sites can be detected through the mobility shift. If the sequences of the probe chains are changed in a controlled manner, the location and base of the mutant can also be determined.     

Molecular dynamics of minimal B‐DNA

Stable and accurate molecular dynamics (MD) of B‐DNA duplexes can be obtained in inexpensive computational conditions where only the minor groove is filled with water while the bulk solvent is represented implicitly. This model system presents significant theoretical as well as practical interest because, due to its simplicity and exceptional computational performance, it can be employed in simulations of very long DNA fragments. To better understand its properties and clarify the physical background of the effects produced by the limited water shell, dynamics of several different DNA oligomers was studied. It is found that optimal simulation conditions are reached when the explicit water is confined within the minor groove while the major groove is cleaned periodically. The internal solvent mobility appears high enough to observe in the nanosecond time scale spontaneous formation of sequence‐specific hydration patterns known from experiments. It is shown that the model produces stable MD trajectories close to the B‐DNA form regardless of the base pair sequence and that, on the other hand, the dynamics are strongly sequence dependent. Independent observations suggest that B‐DNA with only minor groove hydrated resembles its natural thermodynamic state at low water concentration; therefore, this model system can be tentatively called “minimal B‐DNA.” © 2001 John Wiley & Sons, Inc. J Comput Chem 22: 457–467, 2001     

Modification of the electrokinetic properties of reversible electrophoresis gels for the separation and preparation of DNA

The effect of adding linear polymers to a novel reversible electrophoretic was measured. Reversible gels are formed using the polyanionic carbohydrate polymer, gellan gum. Gellan gum forms strong stable gels in the presence of divalent cations or diamines. The gels are reversible (return to solution) by changing the ionic environment or pH. Gellan gum is an anionic polymer, and the electrophoresis gels have considerable electroosmotic flow (EOF) toward the negative electrode. We measured the EOF in gellan gum electrophoresis gels as a function of gel concentration, buffer composition, and linear polymer additive. The linear polymers used in this study were polyethylene oxide and hydroxyethyl cellulose. Both polymers reduced EOF in the gels, in a manner dependent on molecular weight. Polymers with high molecular weight were more effective at reducing EOF. The addition of polymers increased the resolution of low molecular weight DNA. Native gellan gum resolved DNA from approx 50,000 to 1000 bp. Addition of the polymers resolved DNA down to approx 50 bp, in some instances. The influence of the polymers on circular plasmid DNA was also investigated. Addition of high molecular weight polyethylene oxide reduced the electrophoretic mobility of the nicked circular form compared to the supercoiled form.     

Impedance‐Based DNA Biosensor Employing Molecular Beacon DNA as Probe and Thionine as Charge Neutralizer

In this paper, we present an impedance‐based DNA biosensor using thionine intercalation to amplify DNA hybridization signal. Beacon single‐stranded DNA (ssDNA) probe and mercaptoacetic acid were self‐assembled onto a Au electrode by forming Au? S bonds. These beacon ssDNAs were hybridized with the complementary sequences around the loop structure. Then thionine was intercalated into the double‐stranded DNA (dsDNA) immobilized on the Au electrode surface. Due to the neutralization of the negative charges of dsDNA by the intercalated thionine, the electronic transfer resistance (R_et) of the DNA modified Au electrode was significantly diminished. Herein, the decreased value of R_et resulted from the thionine intercalating into dsDNA was employed as the hybridization signal. SDS was used to reduce the unspecific adsorption between ssDNA and thionine. Several experimental conditions, including the surface coverage of ssDNA probe on Au electrode, the hybridization temperature and time were all optimized. Moreover, the hybridization reactions of the unstructured linear ssDNA probe and the structured beacon ssDNA probe with their complementary sequences were compared in this work. The sensitivity of the presented DNA biosensor highlighted that the intercalation of thionine into dsDNA was an efficient approach to amplify the hybridization signal using impedance detection technique. Additionally, in this DNA biosensing protocol, beacon ssDNA has a good ability to distinguish target DNA sequences. This results in a higher specificity than using traditional unstructured DNA probe.     

Sequence‐Dependent Photocurrent Generation through Long‐Distance Excess‐Electron Transfer in DNA

Given its well‐ordered continuous π stacking of nucleobases, DNA has been considered as a biomaterial for charge transfer in biosensors. For cathodic photocurrent generation resulting from hole transfer in DNA, sensitivity to DNA structure and base‐pair stacking has been confirmed. However, such information has not been provided for anodic photocurrent generation resulting from excess‐electron transfer in DNA. In the present study, we measured the anodic photocurrent of a DNA‐modified Au electrode. Our results demonstrate long‐distance excess‐electron transfer in DNA, which is dominated by a hopping mechanism, and the photocurrent generation is sequence dependent.     

Site‐Directed,On‐Surface Assembly of DNA Nanostructures

Two‐dimensional DNA lattices have been assembled from DNA double‐crossover (DX) motifs on DNA‐encoded surfaces in a site‐specific manner. The lattices contained two types of single‐stranded protruding arms pointing into opposite directions of the plane. One type of these protruding arms served to anchor the DNA lattice on the solid support through specific hybridization with surface‐bound, complementary capture oligomers. The other type of arms allowed for further attachment of DNA‐tethered probe molecules on the opposite side of the lattices exposed to the solution. Site‐specific lattice assembly and attachment of fluorophore‐labeled oligonucleotides and DNA–protein conjugates was demonstrated using DNA microarrays on flat, transparent mica substrates. Owing to their programmable orientation and addressability over a broad dynamic range from the nanometer to the millimeter length scale, such supramolecular architecture might be used for presenting biomolecules on surfaces, for instance, in biosensor applications.     

Universal pH‐Responsive and Metal‐Ion‐Free Self‐Assembly of DNA Nanostructures

pH‐responsiveness has been widely pursued in dynamic DNA nanotechnology, owing to its potential in biosensing, controlled release, and nanomachinery. pH‐triggering systems mostly depend on specific designs of DNA sequences. However, sequence‐independent regulation could provide a more general tool to achieve pH‐responsive DNA assembly, which has yet to be developed. Herein, we propose a mechanism for dynamic DNA assembly by utilizing ethylenediamine (EN) as a reversibly chargeable (via protonation) molecule to overcome electrostatic repulsions. This strategy provides a universal pH‐responsivity for DNA assembly since the regulation originates from externally co‐existing EN rather than specific DNA sequences. Furthermore, it endows structural DNA nanotechnology with the benefits of a metal‐ion‐free environment including nuclease resistance. The concept could in principle be expanded to other organic molecules which may bring unique controls to dynamic DNA assembly.     

Reconfigurable T‐junction DNA Origami

DNA self‐assembly allows the construction of nanometre‐scale structures and devices. Structures with thousands of unique components are routinely assembled in good yield. Experimental progress has been rapid, based largely on empirical design rules. Herein, we demonstrate a DNA origami technique designed as a model system with which to explore the mechanism of assembly. The origami fold is controlled through single‐stranded loops embedded in a double‐stranded DNA template and is programmed by a set of double‐stranded linkers that specify pairwise interactions between loop sequences. Assembly is via T‐junctions formed by hybridization of single‐stranded overhangs on the linkers with the loops. The sequence of loops on the template and the set of interaction rules embodied in the linkers can be reconfigured with ease. We show that a set of just two interaction rules can be used to assemble simple T‐junction origami motifs and that assembly can be performed at room temperature.     

DNA Origami Post‐Processing by CRISPR‐Cas12a

Customizable nanostructures built through the DNA‐origami technique hold tremendous promise in nanomaterial fabrication and biotechnology. Despite the cutting‐edge tools for DNA‐origami design and preparation, it remains challenging to separate structural components of an architecture built from—thus held together by—a continuous scaffold strand, which in turn limits the modularity and function of the DNA‐origami devices. To address this challenge, here we present an enzymatic method to clean up and reconfigure DNA‐origami structures. We target single‐stranded (ss) regions of DNA‐origami structures and remove them with CRISPR‐Cas12a, a hyper‐active ssDNA endonuclease without sequence specificity. We demonstrate the utility of this facile, selective post‐processing method on DNA structures with various geometrical and mechanical properties, realizing intricate structures and structural transformations that were previously difficult to engineer. Given the biocompatibility of Cas12a‐like enzymes, this versatile tool may be programmed in the future to operate functional nanodevices in cells.     

Evaluation of DNA bending models in their capacity to predict electrophoretic migration anomalies of satellite DNA sequences

DNA containing a sequence that generates a local curvature exhibits a pronounced retardation in electrophoretic mobility. Various theoretical models have been proposed to explain relationship between DNA structural features and migration anomaly. Here, we studied the capacity of 15 static wedge‐bending models to predict electrophoretic behavior of 69 satellite monomers derived from four divergent families. All monomers exhibited retarded mobility in PAGE corresponding to retardation factors ranging 1.02–1.54. The curvature varied both within and across the groups and correlated with the number, position, and lengths of A‐tracts. Two dinucleotide models provided strong correlation between gel mobility and curvature prediction; two trinucleotide models were satisfactory while remaining dinucleotide models provided intermediate results with reliable prediction for subsets of sequences only. In some cases, similarly shaped molecules exhibited relatively large differences in mobility and vice versa. Generally less accurate predictions were obtained in groups containing less homogeneous sequences possessing distinct structural features. In conclusion, relatively universal theoretical models were identified suitable for the analysis of natural sequences known to harbor relatively moderate curvature. These models could be potentially applied to genome wide studies. However, in silico predictions should be viewed in context of experimental measurement of intrinsic DNA curvature.     

Site‐Directed,On‐Surface Assembly of DNA Nanostructures

Two‐dimensional DNA lattices have been assembled from DNA double‐crossover (DX) motifs on DNA‐encoded surfaces in a site‐specific manner. The lattices contained two types of single‐stranded protruding arms pointing into opposite directions of the plane. One type of these protruding arms served to anchor the DNA lattice on the solid support through specific hybridization with surface‐bound, complementary capture oligomers. The other type of arms allowed for further attachment of DNA‐tethered probe molecules on the opposite side of the lattices exposed to the solution. Site‐specific lattice assembly and attachment of fluorophore‐labeled oligonucleotides and DNA–protein conjugates was demonstrated using DNA microarrays on flat, transparent mica substrates. Owing to their programmable orientation and addressability over a broad dynamic range from the nanometer to the millimeter length scale, such supramolecular architecture might be used for presenting biomolecules on surfaces, for instance, in biosensor applications.     

Determination of DNA Hypermethylation Using Anti‐cancer Drug‐Temozolomide

An electrochemical drug‐DNA biosensor was developed for the detection of interaction between the anti‐cancer drug, Temozolomide (TMZ), and DNA sequences by using Differential Pulse Voltammetry at the graphite electrode surfaces. TMZ is a pro‐drug and an alkylating agent that crosses the blood‐brain barrier, so it is mainly used for brain cancers treatment. In this study, we aim to develop a‐proof‐of‐concept study to investigate the effect of TMZ on formerly methylated DNA sequences since TMZ shows its anti‐cancer activity by methylating the DNA. Interaction between TMZ and DNA causes localized distortion of DNA away from an idealized B‐form, resulting in a wider major groove and greater steric accessibility of functional groups in the base of the groove. According to the results, TMZ behaves as a ‘hybridization indicator’ because of its different electrochemical behavior to different strands of DNA. After interaction with TMZ, hybrid (double stranded DNA‐dsDNA) signals decreased dramatically whereas probe (single stranded DNA‐ssDNA) and control signals remain almost unchanged. The signal differences enabled us to distinguish ssDNA and dsDNA without using a label or tag. It is the first study to demonstrate the interaction between the TMZ and dsDNA created from probe and target. We use specific oligonucleotides sequences instead of using long dsDNA sequences.