DNA Crystals Research Articles

Creation of Metal-Complex-Integrated Tensegrity Triangle DNA Crystals.

Structural DNA nanotechnology is an emerging field and is expected to be used for various applications in materials science. In this study, we designed a DNA tensegrity triangle to accommodate the bipyridine complexes with metal ions (Ni2+ and Fe2+) at the center of the space within the triangle. A metal-bipyridine-incorporated DNA tensegrity triangle was crystalized, and the presence of metals within it was confirmed through X-ray crystal structure analysis. A signal of the anomalous dispersion effect derived from metal was observed in the center of the DNA triangle.

Tuning Chemical DNA Ligation within DNA Crystals and Protein-DNA Cocrystals.

Biomolecular crystals can serve as materials for a plethora of applications including precise guest entrapment. However, as grown, biomolecular crystals are fragile in solutions other than their growth conditions. For crystals to achieve their full potential as hosts for other molecules, crystals can be made stronger with bioconjugation. Building on our previous work using carbodiimide 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) for chemical ligation, here, we investigate DNA junction architecture through sticky base overhang lengths and the role of scaffold proteins in cross-linking within two classes of biomolecular crystals: cocrystals of DNA-binding proteins and pure DNA crystals. Both crystal classes contain DNA junctions where DNA strands stack up end-to-end. Ligation yields were studied as a function of sticky base overhang length and terminal phosphorylation status. The best ligation performance for both crystal classes was achieved with longer sticky overhangs and terminal 3'phosphates. Notably, EDC chemical ligation was achieved in crystals with pore sizes too small for intracrystal transport of ligase enzyme. Postassembly cross-linking produced dramatic stability improvements for both DNA crystals and cocrystals in water and blood serum. The results presented may help crystals containing DNA achieve broader application utility, including as structural biology scaffolds.

Texture identification in liquid crystal-protein droplets using evaporative drying, generalized additive modeling, and K-means Clustering

Sessile drying droplets manifest distinct morphological patterns, encompassing diverse systems, viz., DNA, proteins, blood, and protein-liquid crystal (LC) complexes. This study employs an integrated methodology that combines drying droplet, image texture analysis (features from First Order Statistics, Gray Level Co-occurrence Matrix, Gray Level Run Length Matrix, Gray Level Size Zone Matrix, and Gray Level Dependence Matrix), and statistical data analysis (Generalized Additive Modeling and K-means clustering). It provides a comprehensive qualitative and quantitative exploration by examining LC-protein droplets at varying initial phosphate buffered concentrations (0x, 0.25x, 0.5x, 0.75x, and 1x) during the drying process under optical microscopy with crossed polarizing configuration. Notably, it unveils distinct LC-protein textures across three drying stages: initial, middle, and final. The Generalized Additive Modeling (GAM) reveals that all the features significantly contribute to differentiating LC-protein droplets. Integrating the K-means clustering method with GAM analysis elucidates how textures evolve through the three drying stages compared to the entire drying process. Notably, the final drying stage stands out with well-defined, non-overlapping clusters, supporting the visual observations of unique LC textures. Furthermore, this paper contributes valuable insights, showcasing the efficacy of drying droplets as a rapid and straightforward tool for characterizing and classifying dynamic LC textures.Graphical

Programming crystallization kinetics of self-assembled DNA crystals with 5-methylcytosine modification

Self-assembled DNA crystals offer a precise chemical platform at the ångström-scale for DNA nanotechnology, holding enormous potential in material separation, catalysis, and DNA data storage. However, accurately controlling the crystallization kinetics of such DNA crystals remains challenging. Herein, we found that atomic-level 5-methylcytosine (5mC) modification can regulate the crystallization kinetics of DNA crystal by tuning the hybridization rates of DNA motifs. We discovered that by manipulating the axial and combination of 5mC modification on the sticky ends of DNA tensegrity triangle motifs, we can obtain a series of DNA crystals with controllable morphological features. Through DNA-PAINT and FRET-labeled DNA strand displacement experiments, we elucidate that atomic-level 5mC modification enhances the affinity constant of DNA hybridization at both the single-molecule and macroscopic scales. This enhancement can be harnessed for kinetic-driven control of the preferential growth direction of DNA crystals. The 5mC modification strategy can overcome the limitations of DNA sequence design imposed by limited nucleobase numbers in various DNA hybridization reactions. This strategy provides a new avenue for the manipulation of DNA crystal structure, valuable for the advancement of DNA and biomacromolecular crystallography.

DNA hybrid florescent chrominance sensor: Drug sensing through direct gradient florescent color transformation of hybrid DNA crystals loaded lanthanide (Eu3+/Tb3+) complexes

In this study, a highly sensitive anticancer drug sensor was synthesized using DNA crystals loaded with luminescent Ln3+(Eu3+/Tb3+) complexes. The sensors exhibited a pronounced color transformation property due to the differential quenching of Eu3+ and Tb3+ emissions upon drug interactions. These luminescent hybrid DNA crystals present a significant advancement in sensing anticancer drugs like curcumin, transitioning from monitoring fluorescence intensity changes to a visually discernable color change. This new approach eliminates the need for specialized instrumentation. Owing to the intrinsic biocompatibility of materials composed of DNA, this hybrid sensor has the potential to sense drugs within the human body. Hybrid crystals loaded with a single complex will lose their fluorescence properties due to drug quenching; however, crystals loaded with mixed complexes emit distinct fluorescent colors, such as yellow-green and orange-red, at different drug concentrations. This color transformation allows for the direct determination of drug concentration based on crystal color, making the sensing process more simple and easily observable.

Site-Specific Arrangement and Structure Determination of Minor Groove Binding Molecules in Self-Assembled Three-Dimensional DNA Crystals.

The structural analysis of guest molecules in rationally designed and self-assembling DNA crystals has proven an elusive goal since its conception. Oligonucleotide frameworks provide an especially attractive route toward studying DNA-binding molecules by using three-dimensional lattices with defined sequence and structure. In this work, we site-specifically position a suite of minor groove binding molecules, and solve their structures via X-ray crystallography as a proof-of-principle toward scaffolding larger guest species. Two crystal motifs were used to precisely immobilize the molecules DAPI, Hoechst, and netropsin at defined positions in the lattice, allowing us to control occupancy within the crystal. We also solved the structure of a three-ring imidazole-pyrrole-pyrrole polyamide molecule, which sequence-specifically packs in an antiparallel dimeric arrangement within the minor groove. Finally, we engineered a crystal designed to position both netropsin and the polyamide at two distinct locations within the same lattice. Our work elucidates the design principles for the spatial arrangement of functional guests within lattices and opens new potential opportunities for the use of DNA crystals to display and structurally characterize small molecules, peptides, and ultimately proteins of unknown structure.

Three-Dimensional Modeling of CpG DNA Binding with Matrix Lumican Shows Leucine-Rich Repeat Motif Involvement as in TLR9-CpG DNA Interactions.

Lumican is an extracellular matrix proteoglycan known to regulate toll-like receptor (TLR) signaling in innate immune cells. In experimental settings, lumican suppresses TLR9 signaling by binding to and sequestering its synthetic ligand, CpG-DNA, in non-signal permissive endosomes. However, the molecular details of lumican interactions with CpG-DNA are obscure. Here, the 3-D structure of the 22 base-long CpG-DNA (CpG ODN_2395) bound to lumican or TLR9 were modeled using homology modeling and docking methods. Some of the TLR9-CpG ODN_2395 features predicted by our model are consistent with the previously reported TLR9-CpG DNA crystal structure, substantiating our current analysis. Our modeling indicated a smaller buried surface area for lumican-CpG ODN_2395 (1803 Å2) compared to that of TLR9-CpG ODN_2395 (2094 Å2), implying a potentially lower binding strength for lumican and CpG-DNA than TLR9 and CpG-DNA. The docking analysis identified 32 amino acids in lumican LRR1-11 interacting with CpG ODN_2395, primarily through hydrogen bonding, salt-bridges, and hydrophobic interactions. Our study provides molecular insights into lumican and CpG-DNA interactions that may lead to molecular targets for modulating TLR9-mediated inflammation and autoimmunity.

Structure determination of a DNA crystal by MicroED

Microcrystal electron diffraction (MicroED) is a powerful tool for determining high-resolution structures of microcrystals from a diverse array of biomolecular, chemical, and material samples. In this study, we apply MicroED to DNA crystals, which have not been previously analyzed using this technique. We utilized the d(CGCGCG)2 DNA duplex as a model sample and employed cryo-FIB milling to create thin lamella for diffraction data collection. The MicroED data collection and subsequent processing resulted in a 1.10Å resolution structure of the d(CGCGCG)2 DNA, demonstrating the successful application of cryo-FIB milling and MicroED to the investigation of nucleic acid crystals.

Heterobimetallic Base Pair Programming in Designer 3D DNA Crystals.

Metal-mediated DNA (mmDNA) presents a pathway toward engineering bioinorganic and electronic behavior into DNA devices. Many chemical and biophysical forces drive the programmable chelation of metals between pyrimidine base pairs. Here, we developed a crystallographic method using the three-dimensional (3D) DNA tensegrity triangle motif to capture single- and multi-metal binding modes across granular changes to environmental pH using anomalous scattering. Leveraging this programmable crystal, we determined 28 biomolecular structures to capture mmDNA reactions. We found that silver(I) binds with increasing occupancy in T-T and U-U pairs at elevated pH levels, and we exploited this to capture silver(I) and mercury(II) within the same base pair and to isolate the titration points for homo- and heterometal base pair modes. We additionally determined the structure of a C-C pair with both silver(I) and mercury(II). Finally, we extend our paradigm to capture cadmium(II) in T-T pairs together with mercury(II) at high pH. The precision self-assembly of heterobimetallic DNA chemistry at the sub-nanometer scale will enable atomistic design frameworks for more elaborate mmDNA-based nanodevices and nanotechnologies.

Implementing Logic Gates by DNA Crystal Engineering.

DNA self-assembly computation is attractive for its potential to perform massively parallel information processing at the molecular level while at the same time maintaining its natural biocompatibility. It has been extensively studied at the individual molecule level, but not as much as ensembles in 3D. Here, the feasibility of implementing logic gates, the basic computation operations, in large ensembles: macroscopic, engineered 3D DNA crystals is demonstrated. The building blocks are the recently developed DNA double crossover-like (DXL) motifs. They can associate with each other via sticky-end cohesion. Common logic gates are realized by encoding the inputs within the sticky ends of the motifs. The outputs are demonstrated through the formation of macroscopic crystals that can be easily observed. This study points to a new direction of construction of complex 3D crystal architectures and DNA-based biosensors with easy readouts.

Exploring the Potential of Three-Dimensional DNA Crystals in Nanotechnology: Design, Optimization, and Applications.

DNA has been used as a robust material for the building of a variety of nanoscale structures and devices owing to its unique properties. Structural DNA nanotechnology has reported a wide range of applications including computing, photonics, synthetic biology, biosensing, bioimaging, and therapeutic delivery, among others. Nevertheless, the foundational goal of structural DNA nanotechnology is exploiting DNA molecules to build three-dimensional crystals as periodic molecular scaffolds to precisely align, obtain, or collect desired guest molecules. Over the past 30 years, a series of 3D DNA crystals have been rationally designed and developed. This review aims to showcase various 3D DNA crystals, their design, optimization, applications, and the crystallization conditions utilized. Additionally, the history of nucleic acid crystallography and potential future directions for 3D DNA crystals in the era of nanotechnology are discussed.

Divergence and Convergence: Complexity Emerges in Crystal Engineering from an 8-mer DNA.

Biology provides plenty of examples on achieving complicated structures out of minimal numbers of building blocks. In contrast, structural complexity of designed molecular systems is achieved by increasing the numbers of component molecules. In this study, the component DNA strand assembles into a highly complex crystal structure via an unusual path of divergence and convergence. This assembly path suggests a route to minimalists for increasing structural complexity. The original purpose of this study is to engineer DNA crystals with high resolution, which is the primary motivation and a key objective for structural DNA nanotechnology. Despite great efforts in the last 40 years, engineered DNA crystals have not yet consistently reached resolution better than 2.5 Å, limiting their potential uses. Our research has shown that small, symmetrical building blocks generally lead to high resolution crystals. Herein, by following this principle, we report an engineered DNA crystal with unprecedented high resolution (2.17 Å) assembled from one single DNA component: an 8-base-long DNA strand. This system has three unique characteristics: (1) It has a very complex architecture, (2) the same DNA strand forms two different structural motifs, both of which are incorporated into the final crystal, and (3) the component DNA molecule is only an 8-base-long DNA strand, which is, arguably, the smallest DNA motif for DNA nanostructures to date. This high resolution opens the possibility of using these DNA crystals to precisely organize guest molecules at the Å level, which could stimulate a range of new investigations.

DNA lattice growth with single, double, and triple double-crossover boundaries by stepwise self-assembly

Construction of various nanostructures with nanometre-scale precision through various DNA building blocks depends upon self-assembly, base-pair complementarity and sequence programmability. During annealing, unit tiles are formed by the complementarity of base pairs in each strand. Enhancement of growth of target lattices is expected if seed lattices (i.e. boundaries for growth of target lattices) are initially present in a test tube during annealing. Although most processes for annealing DNA nanostructures adopt a one-step high temperature method, multi-step annealing provides certain advantages such as reusability of unit tiles and tuneability of lattice formation. We can construct target lattices effectively (through multi-step annealing) and efficiently (via boundaries) by multi-step annealing and combining boundaries. Here, we construct efficient boundaries made of single, double, and triple double-crossover DNA tiles for growth of DNA lattices. Two unit double-crossover DNA tile-based lattices and copy-logic implemented algorithmic lattices were introduced to test the growth of target lattices on boundaries. We used multi-step annealing to tune the formation of DNA crystals during fabrication of DNA crystals comprised of boundaries and target lattices. The formation of target DNA lattices was visualized using atomic force microscopy (AFM). The borders between boundaries and lattices in a single crystal were clearly differentiable from AFM images. Our method provides way to construct various types of lattices in a single crystal, which might generate various patterns and enhance the information capacity in a given crystal.

Engineering DNA Crystals toward Studying DNA-Guest Molecule Interactions.

Sequence-selective recognition of DNA duplexes is important for a wide range of applications including regulating gene expression, drug development, and genome editing. Many small molecules can bind DNA duplexes with sequence selectivity. It remains as a challenge how to reliably and conveniently obtain the detailed structural information on DNA-molecule interactions because such information is critically needed for understanding the underlying rules of DNA-molecule interactions. If those rules were understood, we could design molecules to recognize DNA duplexes with a sequence preference and intervene in related biological processes, such as disease treatment. Here, we have demonstrated that DNA crystal engineering is a potential solution. A molecule-binding DNA sequence is engineered to self-assemble into highly ordered DNA crystals. An X-ray crystallographic study of molecule-DNA cocrystals reveals the structural details on how the molecule interacts with the DNA duplex. In this approach, the DNA will serve two functions: (1) being part of the molecule to be studied and (2) forming the crystal lattice. It is conceivable that this method will be a general method for studying drug/peptide-DNA interactions. The resulting DNA crystals may also find use as separation matrices, as hosts for catalysts, and as media for material storage.

The Formation and Displacement of Ordered DNA Triplexes in Self-Assembled Three-Dimensional DNA Crystals.

Reconfigurable structures engineered through DNA hybridization and self-assembly offer both structural and dynamic applications in nanotechnology. Here, we have demonstrated that strand displacement of triplex-forming oligonucleotides (TFOs) can be translated to a robust macroscopic DNA crystal by coloring the crystals with covalently attached fluorescent dyes. We show that three different types of triplex strand displacement are feasible within the DNA crystals and the bound TFOs can be removed and/or replaced by (a) changing the pH from 5 to 7, (b) the addition of the Watson-Crick complement to a TFO containing a short toehold, and (c) the addition of a longer TFO that uses the duplex edge as a toehold. We have also proved by X-ray diffraction that the structure of the crystals remains as designed in the presence of the TFOs.

The Rule of Thirds: Controlling Junction Chirality and Polarity in 3D DNA Tiles.

The successful self-assembly of tensegrity triangle DNA crystals heralded the ability to programmably construct macroscopic crystalline nanomaterials from rationally-designed, nanoscale components. This 3D DNA tile owes its "tensegrity" nature to its three rotationally stacked double helices locked together by the tensile winding of a center strand segmented into 7 base pair (bp) inter-junction regions, corresponding to two-thirds of a helical turn of DNA. All reported tensegrity triangles to date have employed turn inter-junction segments, yielding right-handed, antiparallel, "J1" junctions. Here a minimal DNA triangle motif consisting of 3-bp inter-junction segments, or one-third of a helical turn is reported. It is found that the minimal motif exhibits a reversed morphology with a left-handed tertiary structure mediated by a locally-parallel Holliday junction-the "L1" junction. This parallel junction yields a predicted helical groove matching pattern that breaks the pseudosymmetry between tile faces, and the junction morphology further suggests a folding mechanism. A Rule of Thirds by which supramolecular chirality can be programmed through inter-junction DNA segment length is identified. These results underscore the role that global topological forces play in determining local DNA architecture and ultimately point to an under-explored class of self-assembling, chiral nanomaterials for topological processes in biological systems.

Highly Symmetric, Self-Assembling 3D DNA Crystals with Cubic and Trigonal Lattices.

The rational design of nanoscopic DNA tiles has yielded highly ordered crystalline matter in 2D and 3D. The most well-studied 3D tile is the DNA tensegrity triangle, which is known to self-assemble into macroscopic crystals. However, contemporary rational design parameters for 3D DNA crystals nearly universally invoke integer numbers of DNA helical turns and Watson-Crick (WC) base pairs. In this study, 24-bp edges are substituted into a previously 21-bp (two helical turns of DNA) tensegrity triangle motif to explore whether such unconventional motif can self-assemble into 3D crystals. The use of noncanonical base pairs in the sticky ends results in a cubic arrangement of tensegrity triangles with exceedingly high symmetry, assembling a lattice from winding helical axes and diamond-like tessellation patterns. Reverting this motif to sticky ends with Watson-Crick pairs results in a trigonal hexagonal arrangement, replicating this diamond arrangement in a hexagonal context. These results showcase that the authors can generate unexpected, highly complex, pathways for materials design by testing modifications to 3D tiles without prior knowledge of the ensuing symmetry. This study expands the rational design toolbox for DNA nanotechnology; and it further illustrates the existence of yet-unexplored arrangements of crystalline soft matter.

Структурные особенности кластеров ДНК-DPS при изменении концентрации 4- гексилрезорцина

The formation of complexes and crystals of bacterial nucleoid DNA inside bacterial cells in response to adverse external influences is of great interest both in biophysics and structural biology, and in various fields of industry. The main role in the condensation and crystallization of DNA in bacteria under stressful conditions (starvation, temperature, oxidative, and other types of stress) is played by DNA-binding proteins DPS. In this work, the dynamic behavior and structure of Escherichia coli DPS protein complexes in solution with short-chain DNA (25 base pairs) are studied using classical molecular dynamics in the all-atom approximation with a change in the concentration of the phenolic lipid 4-hexylresorcinol, which is a chemical analog of bacterial anabiosis inducers. The effect of changing the concentration of 
 4-hexylresorcinol from 0 to 50, 100 and 500 molecules on the DNA-DPS complex is being studied. By searching for the linear interaction energy, data were obtained on the free energies of DNA-protein binding without and in the presence of 4-hexylresorcinol. It has been shown that high concentrations of 
 4-hexylresorcinol promote the formation of DNA complexes with DPS. To identify the features of the dynamic behavior of DNA and protein, the principal component analysis was carried out. The spatial and energy characteristics of the complexes was obtained.

Mechanical deformation behaviors and structural properties of ligated DNA crystals

DNA self-assembly has emerged as a powerful strategy for constructing complex nanostructures. While the mechanics of individual DNA strands have been studied extensively, the deformation behaviors and structural properties of self-assembled architectures are not well understood. This is partly due to the small dimensions and limited experimental methods available. DNA crystals are macroscopic crystalline structures assembled from nanoscale motifs via sticky-end association. The large DNA constructs may thus be an ideal platform to study structural mechanics. Here, we investigate the fundamental mechanical properties and behaviors of ligated DNA crystals made of tensegrity triangular motifs. We perform coarse-grained molecular dynamics simulations and confirm the results with nanoindentation experiments using atomic force microscopy. We observe various deformation modes, including untension, linear elasticity, duplex dissociation, and single-stranded component stretch. We find that the mechanical properties of a DNA architecture are correlated with those of its components. However, the structure shows complex behaviors which may not be predicted by components alone and the architectural design must be considered.

The effect of Holliday junction sequence and isomeric form on the self-assembly of rationally designed DNA crystals

The effect of Holliday junction sequence and isomeric form on the self-assembly of rationally designed DNA crystals