Complementary Sticky Ends Research Articles

Slow Freeze‐Thaw Cycles Enhanced Hybridization of Kilobase DNA with Long Complementary Sticky Ends

AbstractThe creation of large information molecules may have played an essential role in the origins of life. In this study, we conducted slow freeze‐thaw (F/T) experiments to test the possibility of enhanced hybridization between the complementary sticky ends attached to kilobase‐sized DNA fragments at sub‐nanomolar concentrations. DNA fragments of 2‐ and 3‐kilobase pairs (kbp) with 50‐base complementary sticky ends that can form 5 kbp‐sized hybridization products were mixed. While simple incubation provided little hybridization product, significantly effective hybridization was observed after freezing and thawing at a controlled time rate (<0.3 K min−1), even with small DNA concentrations (<1 nM). Furthermore, slow thawing had a more effect on hybridization than slow freezing. The reaction efficiency was reduced by rapid thawing instead of slow thawing, suggesting that the eutectic phase concentration played an important role in hybridization. A slow F/T cycle was effective even for the hybridization reaction between two 10 kbp DNA fragments, which yielded a 20 kbp product at sub‐nanomolar concentrations. Repeating the slow F/T cycle significantly improved the reaction efficiency. The possible role of the F/T cycles in early Earth environments is discussed here.

A Self‐Assembled Rhombohedral DNA Crystal Scaffold with Tunable Cavity Sizes and High‐Resolution Structural Detail

AbstractDNA is an ideal molecule for the construction of 3D crystals with tunable properties owing to its high programmability based on canonical Watson–Crick base pairing, with crystal assembly in all three dimensions facilitated by immobile Holliday junctions and sticky end cohesion. Despite the promise of these systems, only a handful of unique crystal scaffolds have been reported. Herein, we describe a new crystal system with a repeating sequence that mediates the assembly of a 3D scaffold via a series of Holliday junctions linked together with complementary sticky ends. By using an optimized junction sequence, we could determine a high‐resolution (2.7 Å) structure containing R3 crystal symmetry, with a slight subsequent improvement (2.6 Å) using a modified sticky‐end sequence. The immobile Holliday junction sequence allowed us to produce crystals that provided unprecedented atomic detail. In addition, we expanded the crystal cavities by 50 % by adding an additional helical turn between junctions, and we solved the structure to 4.5 Å resolution by molecular replacement.

A Self-Assembled Rhombohedral DNA Crystal Scaffold with Tunable Cavity Sizes and High-Resolution Structural Detail.

DNA is an ideal molecule for the construction of 3D crystals with tunable properties owing to its high programmability based on canonical Watson-Crick base pairing, with crystal assembly in all three dimensions facilitated by immobile Holliday junctions and sticky end cohesion. Despite the promise of these systems, only a handful of unique crystal scaffolds have been reported. Herein, we describe a new crystal system with a repeating sequence that mediates the assembly of a 3D scaffold via a series of Holliday junctions linked together with complementary sticky ends. By using an optimized junction sequence, we could determine a high-resolution (2.7 Å) structure containing R3 crystal symmetry, with a slight subsequent improvement (2.6 Å) using a modified sticky-end sequence. The immobile Holliday junction sequence allowed us to produce crystals that provided unprecedented atomic detail. In addition, we expanded the crystal cavities by 50 % by adding an additional helical turn between junctions, and we solved the structure to 4.5 Å resolution by molecular replacement.

PH-Responsive and Gemcitabine-Containing DNA Nanogel To Facilitate the Chemodrug Delivery

Herein, we construct a structure-switchable gemcitabine (Ge)-containing DNA nanogel that can respond to the intracellular acidic environment, subsequently facilitating the chemodrug release inside the cells. Based on the structural similarity between Ge and deoxycytidine (dC), dC nucleotides in the component DNA strands used for nanogel assembly are fully replaced by Ge during their synthesis. By changing the designed sequences, two Ge-containing Y-shaped motifs with different sticky ends are first assembled and then associated together to form nanogel by sticky-end hybridizations. In particular, one of the sticky-end sequences is arbitrarily designed to be rich of Ge and the other is designed to be partially complementary to the first Ge-rich sticky end. At the neutral or basic condition, the Ge-rich sticky ends hybridize with the partially complementary sticky ends on the second Y motifs, keeping the assembled nanogel stable. Upon being exposed to the acidic condition, Ge-rich sticky ends intend to form intramolecular i-motif-like quadruplex structures, resulting in the disassembly of the nanogel. On the one hand, the nanosized feature enables the Ge-containing nanogel with rapid cellular uptake behavior. On the other hand, the pH-responsive feature endows the rapid disassembly of the nanogel to facilitate the enzymatic drug release inside the cell, resulting in the enhanced anticancer activity of the DNA-based drug delivery system.

Ultrasensitive Detection of Serum MicroRNA Using Branched DNA-Based SERS Platform Combining Simultaneous Detection of α-Fetoprotein for Early Diagnosis of Liver Cancer

We provided an ultrasensitive sensing strategy for microRNA detection by first employing branched DNA. With the aid of microcontact printing, we realized the multiplex sensing of different kinds of liver cancer biomarkers: microRNA and protein simultaneously. Delicately designed branched DNA included multiple complementary sticky ends as probe to microRNA capture and the double-stranded rigid branched core to increase the active sticky-ends distance and expose more DNA probes for sensitivity. The branched DNA enables 2 orders of magnitude increase in sensitivity for microRNA detection over single-stranded DNA. The limit of detection reaches as low as 10 attomolar (S/N = 3) for miR-223 and 10-12 M for α-fetoprotein. In addition, this system shows high selectivity and appropriate reproducibility (the relative standard deviation is less than 20%) in physiological media. Serum samples are tested and the results of α-fetoprotein are in good agreement with the current gold-standard method, electrochemiluminescence immunoassay analyzer. The results suggest the reliability of this approach in physiological media and show high potential in the sensing of low abundant microRNA in serum, especially for early diagnosis of primary liver cancers.

DNA cytoskeleton for stabilizing artificial cells

Cell-sized liposomes and droplets coated with lipid layers have been used as platforms for understanding live cells, constructing artificial cells, and implementing functional biomedical tools such as biosensing platforms and drug delivery systems. However, these systems are very fragile, which results from the absence of cytoskeletons in these systems. Here, we construct an artificial cytoskeleton using DNA nanostructures. The designed DNA oligomers form a Y-shaped nanostructure and connect to each other with their complementary sticky ends to form networks. To undercoat lipid membranes with this DNA network, we used cationic lipids that attract negatively charged DNA. By encapsulating the DNA into the droplets, we successfully created a DNA shell underneath the membrane. The DNA shells increased interfacial tension, elastic modulus, and shear modulus of the droplet surface, consequently stabilizing the lipid droplets. Such drastic changes in stability were detected only when the DNA shell was in the gel phase. Furthermore, we demonstrate that liposomes with the DNA gel shell are substantially tolerant against outer osmotic shock. These results clearly show the DNA gel shell is a stabilizer of the lipid membrane akin to the cytoskeleton in live cells.

Protocols for C-Brick DNA Standard Assembly Using Cpf1.

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this characteristic, Cpf1 has been used for the establishment of a DNA assembly standard called C-Brick, which has the advantage of long recognition sites and short scars. On a standard C-Brick vector, there are four Cpf1 recognition sites - the prefix (T1 and T2 sites) and the suffix (T3 and T4 sites) - flanking biological DNA parts. The cleavage of T2 and T3 sites produces complementary sticky ends, which allow for the assembly of DNA parts with T2 and T3 sites. Meanwhile, a short "GGATCC" scar is generated between parts after assembly. As the newly formed plasmid once again contains the four Cpf1 cleavage sites, the method allows for the iterative assembly of DNA parts, which is similar to those of BioBrick and BglBrick standards. A procedure outlining the use of the C-Brick standard to assemble DNA parts is described here. The C-Brick standard can be widely used by scientists, graduate and undergraduate students, and even amateurs.

DNA self-assembly-based fabrication of metallic nanostructures and related nanophotonics

Nanophotonics focuses on the study of the behavior of light and the interaction between light and matter on a nanometer scale. It has often involved metallic nanostructures which can concentrate and guide the light beyond the diffraction limit due to the unique surface plasmons (SPs). Manipulation of light can be accomplished through controlling the morphologies and components of metallic nanostructures to incur special surface plasmons. However, it is still a severe challenge to achieve exquisite control over the morphologies or components of metallic nanostructures: chemical methods can provide anisotropic but highly symmetric metallic nanostructures; lithographic methods have a limited resolution, especially for three-dimensional metallic nanostructures. By comparison, DNA self-assembly-based fabrication of metallic nanostructures is not restricted to these confinements. With the high-fidelity Waston-Crick base pairing, DNA can self-assemble into arbitrary shapes ranging from the simplest double strands to the most sophisticated DNA origami. Due to the electrostatic interactions between negatively charged phosphate backbones and positively charged metal ions, DNA of any shapes can affect the metal ions or atoms to a certain degree. Depending on the shape and base, DNA self-assembly nanostructures can exert different influences on the growth of metallic nanoparticles, which in turn gives rise to deliberately controllable metallic nanostructures. Besides, DNA self-assembly nanostructures can act as ideal templates for the organization of metallic nanoparticles to construct special metallic nanostructures. In this case, DNA-modified metallic nanoparticles are immobilized on DNA self-assembly nanostructures carrying complementary sticky ends. The geometry and component arrangements of metallic nanostructures both can be precisely dictated on the DNA nanostructures by programming the sticky end arrays. Complicated metallic nanostructures which can be hardly fabricated with conventional chemical or lithographic methods have been readily prepared with the DNA self-assembly-based fabrication method, thereby greatly promoting the development of nanophotonics. Therefore, the studies of DNA self-assembly-based fabrication of metallic nanostructures and related nanophotonics have received rapidly growing attention in recent years. This review first gives a brief introduction of the mechanism for breaking the diffraction limit of light with metallic nanostructures based on SPs. Then we give a systematic review on DNA self-assembly-based fabrication of metallic nanostructures and related nanophotonics, which is divided into several parts according to the different pathways by which DNA self-assembly can influence the morphologies or components of metallic nanostructures. Finally, the remaining problems and limitations for the existing DNA self-assembly-based fabrication of metallic nanostructures are presented and an outlook on the future trend of the field is given as well.

Equilibrium gels of trivalent DNA-nanostars: Effect of the ionic strength on the dynamics.

Self-assembling DNA-nanostars are ideal candidates to explore equilibrium gelation in systems composed of limited-valence particles. We present here a light scattering study of the dynamics in a trivalent DNA-nanostars equilibrium gel and of its dependence on ionic strength and concentration. Reversible bonds between different nanostars, whose formation is sensitively dependent on temperature, concentration and ionic strength, are provided by complementary DNA sticky ends. We find that the decay of the density correlations is described by a two-step relaxation process characterised by: i) a slow time scale that varies over nearly four orders of magnitude in a temperature window of less than 30 degrees; ii) an increase of the amplitude (the so-called non-ergodicity factor) of the slow relaxation. The slow process follows an Arrhenius law with temperature. We observe that the activation enthalpy does not depend on the ionic strength and that the dependence of the relaxation time on the ionic strength can be rationalized in terms of the free-energy cost of forming a sticky-end duplex. Finally, we observe that dynamics is insensitive to nanostar concentration, in full agreement with the predicted behaviour in equilibrium gels.

DNAやペプチドの自己集合によるナノバイオ分子システムの構築

In biological system, various nano–assemblies are formed by self–assembly of proteins with high symmetric ternary structures. For example, dodecahedral internal skeleton of tomato bushy stunt virus is self–assembled from C3–symmetric β–sheet subunits. Inspired by such self–assembly process, we have developed a novel strategy for construction of spherical assemblies consisting of biomacromolecules such as DNA and peptides. DNA three–way junctions bearing self–complementary sticky–ends were self–assembled into nanometer to micrometer sized spherical structures depending on the concentration. The three–way component design was extended to the design of the artificial C3–symmetric conjugate which contains three β–sheet–forming peptides FKFEFKFE. Radial–type Trigonal–FKFE self–assembled into nanocapsules with the size of 20 nm. In contrast, wheel–type Trigonal–FKFE self–assembled into nanofibers with uniform width (3 nm). Recently, we designed T–shaped C3–symmetric peptide conjugate Trigonal–glutathiones (TG). TG self–assembled into soft nanospheres with the size of about 100—250 nm, whose size was little affected by the concentration.

From molecular to macroscopic via the rational design of a self-assembled 3D DNA crystal

We live in a macroscopic three-dimensional world, but our best description of the structure of matter is at the atomic and molecular scale. Understanding the relationship between the two scales requires that we bridge from the molecular world to the macroscopic world. Connecting these two domains with atomic precision is a central goal of the natural sciences, but it requires high spatial control of the 3D structure of matter.1 The simplest practical route to producing precisely designed 3D macroscopic objects is to form a crystalline arrangement by self-assembly, because such a periodic array has only conceptually simple requirements: [1] A motif whose 3D structure is robust, [2] dominant affinity interactions between parts of the motif when it self-associates, and [3] a predictable structures for these affinity interactions. Fulfilling all these criteria to produce a 3D periodic system is not easy, but it should readily be achieved by well-structured branched DNA motifs tailed by sticky ends.2 Complementary sticky ends associate with each other preferentially and assume the well-known B-DNA structure when they do so;3 the helically repeating nature of DNA facilitates the construction of a periodic array. It is key that the directions of propagation associated with the sticky ends not share the same plane, but extend to form a 3D arrangement of matter. Here, we report the crystal structure at 4 Å resolution of a designed, self-assembled, 3D crystal based on the DNA tensegrity triangle.4 The data demonstrate clearly that it is possible to design and self-assemble a well-ordered macromolecular 3D crystalline lattice with precise control.

Evaluation of small ligand–protein interaction by ligation reaction with DNA-modified ligand

A method for the evaluation of interactions between protein and ligand using DNA-modified ligands, including signal enhancement of the DNA ligation reactions, is described. For proof of principle, a DNA probe modified by biotin was used. Two DNA probes were prepared with complementary sticky-ends. While one DNA probe was modified at the 5'-end of the sticky-end, the other was not modified. The probes could be ligated together by T4 DNA ligase along the strand without biotin modification. However, in the presence of streptavidin or anti-biotin Fab, the ligation reaction joining the two probes could not occur on either strand.

Ligation of DNA Triangles Containing Double Crossover Molecules

An antiparallel DNA double crossover molecule has been incorporated into one edge of a DNA triangle. The goal in this work is to combine a rigid geometrical motif, the triangle, with a rigid DNA motif, the double crossover, incorporating the properties of branched DNA. The triangle has been designed to contain 3 turns of DNA in each edge. The double crossover molecule produces an extra domain along its edge. The extra domain can be restricted by Bbs I to produce two complementary sticky ends separated by 4.5 turns of DNA. Ligation-closure experiments performed on the restricted triangle yield very long reporter strands and no indications of cyclization, suggesting that the double crossover molecule retains its stiffness when incorporated in the triangle. These experiments are evaluated through the use of denaturing gel electrophoresis. The ligation products have been visualized by atomic force microscopy, under nondenaturing conditions. Clear zigzag species can be identified as triangles ligated in one dimension. The images show no systematic distortions such as permanent bends. These visualizations have been confirmed by alternating triangles with simple double crossover molecules. When double crossover molecules containing 4.0 turns are alternated with triangles, the spacing of the vertexes is increased appropriately. When the double crossover molecules contain 4.5 turns, all the vertexes appear on the same side of the linear fragment, confirming the correlation between chemical input and mesoscopic images in the atomic force microscope. We conclude that double crossover molecules can be incorporated into at least one side of a triangle without any apparent effect on their rigidity.

Rapid and efficient cloning of Alu-PCR products using uracil DNA glycosylase.

By incorporating dUMP residues into the 5' end of PCR primers, one can generate products which, after treatment with uracil DNA glycosylase (UDG), contain 3' overhangs. These overhangs can be annealed to vector molecules with complementary overhangs generated in a similar fashion and transformed directly into Escherichia coli without the need for ligase. We have tested this method of ligation-independent cloning by using UDG to create complementary single-stranded sticky ends between vector and Alu-PCR products generated from cosmid clones containing DNA from human chromosome 21. Using a single primer, Alu-PCR amplifies the sequence between appropriately oriented, repetitive (Alu) sequences in human DNA that are no more than 2 to 3 kb apart. Nineteen Alu-PCR products were observed in four human chromosome 21 cosmids. Thirteen of these products were detected among 48 subclones picked at random after cloning of the Alu-PCR products using UDG. The size or abundance of an Alu-PCR product did not appear to affect significantly the efficiency of cloning. Eight of the subclones were tested and all hybridized to human chromosome 21 DNA. UDG cloning should prove to be a general PCR cloning method that allows one to rapidly subclone small fragments from human genomic DNA.

Modification of DNA ends can decrease end joining relative to homologous recombination in mammalian cells.

In animal cells, exogenous DNA recombines into random chromosomal sites much more frequently than it recombines into homologous sites. Free DNA ends are "recombinogenic" in both processes. To test the effects of specific ends on analogous extrachromosomal processes, we constructed a linear genome of simian virus 40 with terminal repeated sequences. After transfection into monkey cells, the model substrate can circularize by end joining (analogous to random integration) or by homologous recombination between its terminal repeats (analogous to targeted recombination). Since the two types of recombination are in competition with one another, the ratio of homologous-recombination to end-join products is a sensitive indicator of the differential effects of specific ends. Substrates with blunt ends, complementary sticky ends, or mismatched ends generated the same ratio of homologous-recombination to end-join products. However, addition of dideoxynucleotides to the 3' hydroxyls of the substrate decreased the frequency of end joining by a factor of 5-6 relative to homologous recombination. Thus, the frequency of end joining can be decreased relative to that of homologous recombination by modification of the ends of the input DNA. These results suggest an approach to altering the ratio of random to targeted integration in mammalian cells.

Optimization of conditions for the in vitro formation of hybrid DNA molecules by DNA ligase

Conditions for the ligation with T4 induced DNA ligase of two DNA molecules via their complementary sticky ends have been established which lead preferentially to the formation of hybrid molecules. This is demonstrated with two combinations of parent molecules varying greatly in their relative molecular weights. In one case the intact hybrid molecule could be directly isolated. In addition a DNA dependent quantitative electrophoretic assay for DNA ligase activity is described which does not need a radioactively labeled substrate. The ligation procedure has been shown to be useful in molecular cloning experiments.