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A three-dimensional DNA hydrogel was generated by self-assembly of short linear double-stranded DNA (dsDNA) building blocks equipped with sticky ends. The resulting DNA hydrogel is thermoresponsive and the length of the supramolecular dsDNA structures varies with temperature. The average diffusion coefficients of the supramolecular dsDNA structures formed by self-assembly were determined by diffusion-ordered NMR spectroscopy (DOSY NMR) for temperatures higher than 60 °C. Temperature-dependent rheological measurements revealed a gel point of 42±1 °C. Below this temperature, the resulting material behaved as a true gel of high viscosity with values for the storage modulus G' being significantly larger than that for the loss modulus G''. Frequency-dependent rheological measurements at 20 °C revealed a mesh size (ξ) of 15 nm. AFM analysis of the diluted hydrogel in the dry state showed densely packed structures of entangled chains, which are also expected to contain multiple interlocked rings and catenanes.

The linear, double-helical structure of DNA was initially recognized as beautiful, as well as being informative about the mechanism of heredity. Recently, branched DNA molecules have been used to produce nanoscale objects, crystals and machines, all the products of a new field: structural DNA nanotechnology. The inspiration for much of this work has been art, starting from the notion that Escher's woodcut Depth was analogous to a molecular crystal of branched DNA. The article describes how connecting branched molecules together with the “sticky ends” used by genetic engineers has led to 3D crystals, and how Dalí's Butterfly Landscape illuminates the relationship between wrappings of DNA and the crossings in knots or links. Disparate aesthetic patterns are related to branched DNA motifs and constructions.

DNA is a very useful molecule for the programmed self-assembly of 2D and 3D nanoscale objects.[1] The design of these structures exploits Watson–Crick hybridization and strand exchange to stitch linear duplexes into finite assemblies.[2–4] The dimensions of these complexes can be increased by over five orders of magnitude through self-assembly of cohesive single-stranded segments (sticky ends).[5,6] Methods that exploit the sequence addressability of DNA nanostructures will enable the programmable positioning of components in 2D and 3D space, offering applications such as the organization of nanoelectronics,[7] the direction of biological cascades,[8] and the structure determination of periodically positioned molecules by X-ray diffraction.[9] To this end we present a macroscopic 3D crystal based on the 3-fold rotationally symmetric tensegrity triangle[3,6] that can be functionalized by a triplex-forming oligonucleotide on each of its helical edges.

With its ever-increasing viral genetic diversity, accurate diagnosis of porcine reproductive and respiratory syndrome virus (PRRSV) infection is indispensable for PRRSV control. Here, a sensitive graphene oxide (GO)-based FRET method was developed to detect PRRSV-2 based on the ability of GO to quench fluorophore by fluorescence resonance energy transfer (FRET). Using primers and a fluorophore-labeled ssDNA probe targeting a conserved region between the PRRSV M gene and 3'UTR, asymmetric PCR specifically amplified viral ssDNA that could anneal with probe to generate dsDNA only in the presence of virus. Upon exonuclease III treatment to release the probe fluorophore, which degrades dsDNA with blunt ends or recessed 3´-termini, the ssDNA annealed with other probe to generate enhanced fluorescence. This GO-based FRET assay specifically detected both classical and highly pathogenic PRRSV, with analytical sensitivity approaching 10 copies/μL, similar to that of real-time PCR but greater than that of conventional reverse transcription PCR (RT-PCR). Consistent with real-time RT-PCR detection, the assay developed here exhibited high diagnostic sensitivity for virus detection of sera from experimentally and naturally infected pigs. Thus, this novel GO-based FRET assay combined with asymmetric PCR detection is sensitive and specific and will be valuable for future PRRSV diagnosis.

Surface functionalization with bioinspired binding groups is increasingly used to steer nano- and microscale self-assembly processes, with complementary DNA "sticky ends" as one of the most notable examples. The fabrication of well-organized structures is complicated, however, by the sharp association/dissociation transitions and the slow rearrangement kinetics intrinsic to collections of discrete, surface-immobilized binding groups and is aggravated by natural nonuniformities in the surface coating. Here, we demonstrate a novel system of solid microparticles functionalized with specific binding groups-in this case DNA linkers-that are fully mobile along the particle surface. These colloids display qualitatively new behavior and circumvent many of the commonly encountered issues. Importantly, the association/dissociation transition, and thereby the temperature window for equilibrium self-assembly, is much broader. We further find that the linkers are uniformly distributed above the DNA melting temperature, while visibly accumulating at the interparticle contacts below this temperature. The unique combination of binding group mobility with nondeformability, monodispersity, and facile manipulation of solid particles should have a profound impact on DNA-mediated and other bioinspired self-assembly approaches. Moreover, our highly tunable experimental system enables detailed model investigations that will also deepen our fundamental understanding of other systems with surface-mobile binding groups, for instance, biological ligand-receptor interactions.

Plasmonic circular dichroism (CD) responses of hybrid nanostructures containing noble metal nanoparticles and chiral molecules have received increasing interest with various applications in nanophotonics. Chiral biomolecules show strong CD signals typically found in the ultraviolet region, whereas, in the visible range, they produce a weak signal. Strengthening the CD signal in the visible region is of high importance, which could be achieved through fabrication of novel hybrid nanostructures. Herein, gold nanoparticles (GNPs) have been assembled via DNA linker to investigate the possibility of enhancing plasmonic CD signal in the visible range. DNA-linked assemblies with pre- and postannealed conditions were characterized by ultraviolet–visible spectroscopy, dynamic light scattering (DLS), and CD spectropolarimetry. In the presence of DNA linker with sticky ends, the aggregation phenomenon was traced by red shifts of surface plasmon resonance of nanoparticles. Time-dependent hybridization of single-stranded “sticky ends” with DNA-conjugated GNPs and increased probability of hydrogen bond formation lead to enhancement of CD signals in the ultraviolet region. Complexation of biomolecule and nanoparticle assemblies induced enhanced CD signals in the visible range, which was noticed both before and after purification. DLS characterization of the assemblies also confirmed the difference in the size of aggregates, which could be controlled by the linker molecules. This investigation encourages possibility of utilizing plasmonic CD technique as a tool for tracing fabricated nanostructure assemblies with enhanced characterization possibility.

Stable contacts between the regions of nonhomologous chromosomes have long been known from earlycytogenetic studies [1–5]; however, their nature is stillnot sufficiently studied. In recent years, new techniques specifically designed for a more accurate analysis of threedimensional structures of the chromosomes in the nucleus and to identify chromosomalregions that are close enough to each other were developed [6, 7]. These methods ar e based on the fixation ofchromatin structures by treatment with formaldehyde,Cleavage of DNA with an appropriate restrictionenzyme, and subsequent ligation of the sticky ends ofadjacent DNA fragments. Elegant methods for subsequent truncation of recombinant DNA molecules,their amplification by PCR, and analysis of the products by deep sequencing were proposed. Thus, it ispossible to reveal all the possible interactions of aselected region of the genome with other areas.Previously, we have obtained data indicating thatthe expression of

A novel, high sensitive, and specific DNA assay based on gold nanoparticle (AuNP) colorimetric detection and hybridization chain reaction (HCR) amplification has been demonstrated in this article. Two hairpin auxiliary probes were designed with single-stranded DNA (ssDNA) sticky ends which stabilize AuNPs and effectively prevent them from salt-induced aggregation. The target DNA hybridized with the hairpin auxiliary probes and triggered the formation of extended double-stranded DNA (dsDNA) polymers through HCR. As a result, the formed dsDNA polymers provide less stabilization without ssDNA sticky ends, and AuNPs undergo aggregation when salt concentration is increased. Subsequently, a pale purple-to-blue color variation is observed in the colloid solution. The system is simple in design and convenient in operation. The novel strategy eliminates the need for enzymatic reactions, separation processes, chemical modifications, and sophisticated instrumentation. The detection and discrimination process can be seen with the naked eye. The detection limit of this method is lower than or at least comparable to previous AuNP-based methods. Importantly, the protocol offers high selectivity for the determination between perfectly matched target oligonucleotides and targets with single base-pair mismatches.

Interlock is a structural element in DNA G-quadruplexes that can be compared with the commonly used complementary binding of ‘sticky ends’ in DNA duplexes. G-quadruplex interlocking can be a basis for the assembly of higher-order structures. In this study, we formulated a rule to engineer (3+1) interlocked dimeric G-quadruplexes and established the folding topology of the designed DNA sequences by nuclear magnetic resonance spectroscopy. These interlocked G-quadruplexes are very stable and can serve as compact robust scaffolds for various applications. Different structural elements can be engineered in these robust scaffolds. We demonstrated the anti-HIV inhibition activity of the newly designed DNA sequences. Published version

Mycobacteria exploit nonhomologous end-joining (NHEJ) to repair DNA double-strand breaks. The core NHEJ machinery comprises the homodimeric DNA end-binding protein Ku and DNA ligase D (LigD), a modular enzyme composed of a C-terminal ATP-dependent ligase domain (LIG), a central 3'-phosphoesterase domain (PE), and an N-terminal polymerase domain (POL). LigD POL is proficient at adding templated and nontemplated deoxynucleotides and ribonucleotides to DNA ends in vitro and is the catalyst in vivo of unfaithful NHEJ events involving nontemplated single-nucleotide additions to blunt DSB ends. Here, we identify two mycobacterial proteins, PolD1 and PolD2, as stand-alone homologues of the LigD POL domain. Biochemical characterization of PolD1 and PolD2 shows that they resemble LigD POL in their monomeric quaternary structures, their ability to add templated and nontemplated nucleotides to primer-templates and blunt ends, and their preference for rNTPs versus dNTPs. Deletion of polD1, polD2, or both from a Mycobacterium smegmatis strain carrying an inactivating mutation in LigD POL failed to reveal a role for PolD1 or PolD2 in templated nucleotide additions during NHEJ of 5'-overhang DSBs or in clastogen resistance. Whereas our results document the existence and characteristics of new stand-alone members of the LigD POL family of RNA/DNA polymerases, they imply that other polymerases can perform fill-in synthesis during mycobacterial NHEJ.

A new type of DNA grafted polypeptide molecular brush was synthesized via a combination of ring-opening polymerization (ROP) and click chemistry. This conjugation method provides an easy and efficient approach to obtain a hybrid DNA-grafted polypeptide molecular bottlebrush. The structure and assembly behaviors of this hybrid brush were investigated using electrophoresis, UV–vis spectroscopy, transmission electron microscopy (TEM), and atomic force microscopy (AFM). Hierarchical supramolecular assemblies can be obtained through hybridization of two kinds of polypeptide-g-DNA molecular bottlebrushes containing complementary DNA side chains. We further demonstrated that such polypeptide-g-DNA can be hybridized with ds-DNA and DNA-grafted gold nanoparticles to form a supermolecular bottlebrush and hybrid bottlebrush, respectively. In addition, DNA-polypeptide hydrogel can be prepared by hybridization of polypeptide-g-DNA with a linker-ds-DNA, which contains the complementary “sticky ends” to serve as cross-l...

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: a motif that has a robust 3D structure, dominant affinity interactions between parts of the motif when it self-associates, and predictable structures for these affinity interactions. Fulfilling these three criteria to produce a 3D periodic system is not easy, but should readily be achieved with well-structured branched DNA motifs tailed by sticky ends (Zheng et al., Nature 461:74-77, 2009). Herein, a brief introduction to designed 3D DNA crystals from tensegrity triangle is presented.

As Cpf1 cleaves double-stranded DNA in a staggered way, it can be used in DNA assembly. However, the Cpf1 cleavage was found to be inaccurate, which may cause errors in DNA assembly. Here, the Cpf1 cleavage sites were precisely characterized, where the cleavage site on the target strand was around the 22nd base relative to the protospacer adjacent motif site, but the cleavage on the non-target strand was affected by the spacer length. When the spacer length was 20 nt or longer, Cpf1 mainly cleaved around the 14th and the 18th bases on the non-target strand; otherwise, with a shorter spacer (i.e. 17–19 nt), Cpf1 mainly cleaved after the 14th base, generating 8-nt sticky ends. With this finding, Cpf1 with a 17-nt spacer crRNA were employed for in vitro substitution of the actII-orf4 promoter in the actinorhodin biosynthetic cluster with a constitutively expressing promoter. The engineered cluster yielded more actinorhodin and produced actinorhodin from an earlier phase. Moreover, Taq DNA ligase was further employed to increase both the ligation efficiency and the ligation accuracy of the method. We expect this CCTL (Cpf1-assisted Cutting and Taq DNA ligase-mediated Ligation) method can be widely used in in vitro editing of large DNA constructs.

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell's DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Dumbbell probe (DP) attracts increasing interests in rolling circle amplification (RCA). A universal DP production method through cleavage-ligation of hairpin was proposed and optimized. The production is characterized by restriction endonuclease (RE)-induced cleavage ends ligation. It has the advantage of phosphorylation-free, splint-free and purification-free. To optimize designing, we found that the position of RE cleavage sequence in the stem and the primer position in the loop affected the formation and amplification of DP obviously. Both sticky and blunt ends cleaved by RE produce DP efficiently. Moreover, we introduced this DP into circle to circle (C2C) RCA based on the same cleavage-ligation principle and acquired high sensitivity. By combining a two-ligation design and the C2C strategy, specificity for detecting let-7 family members was increased extremely. Furthermore, coreaction of different steps facilitated convenient formation and amplification process of DP.

The goal of this protocol is to allow for the rapid verification of bioinformatically identified terminators. Further, the plasmid (pGR-Blue) is designed specifically for this protocol and allows for the quantification of terminator efficiency. As a proof of concept, six terminators were bioinformatically identified in the mycobacteriophage Bernal13. Once identified, terminators were then made as oligonucleotides with the appropriate sticky ends and annealed together. Using Golden Gate Assembly (GGA), terminators were then cloned into pGR-Blue. Under visible light, false positive colonies appear blue and positively transformed colonies are white/yellow. After induction of an arabinose inducible promoter (pBad) with arabinose, colony strength can be determined by measuring the ratio of green fluorescent protein (GFP) produced to red fluorescent protein (RFP) produced. With pGR-Blue, the protocol can be completed in as little as three days and is ideal in an educational setting. Additionally, results show that this protocol is useful as a means for understanding in silico predictions of terminator efficiency related to the regulation of transcription.

To develop a convenient and sensitive point-of-care test for detecting gene mutations based on allele-specific PCR. To develop a lateral flow strip for visual detection of K-ras mutations based on a modified PCR, a specific DNA tag was covalently linked to the 5′-end of each primer by a nine-carbon linker to produce a sticky end. One of the sticky ends of the PCR products bound to gold nano-particles, while the other sticky end was captured onto a nitrocellulose membrane of lateral flow strips. The lateral flow strip showed a great sensitivity, which detected mutations in as low as 10 tumor cells. The positive rate and accuracy of the lateral flow strip for blood samples were over 92 and 96 %, respectively. The lateral flow strip provides an easy method for sensitive detection of gene mutations based on allele specific-PCR.

Interlocked DNA architectures are useful for DNA nanotechnology because of their mechanically bonded components, which can move relative to one another without disassembling. We describe the design, synthesis, and characterization of novel single-stranded tile (SST) stoppers for the assembly of interlocked DNA architectures. SST stoppers are shown to self-assemble into a square-shaped rigid structure upon mixing 97 oligodeoxynucleotide (ODN) strands. The structures are equipped with a sticky end that is designed for hybridization with the sticky ends of a dsDNA axle of a DNA rotaxane. Because the diameter of the macrocycle threaded onto the axle is 14 nm, the dimension of the square-shaped stopper was designed to be bulky enough to prevent the dethreading of the macrocycle. An asymmetric rotaxane with a SST- and a ring-shaped stopper featuring two stations for hybridization of the macrocycle to the axle was assembled. The macrocycle can be directed towards one or the other station upon triggering with fuel ODNs.

INTRODUCTION In DNA double helices, hydrogen bonds connect the base pairs across the two strands, and stacking bonds act along the helical axis between neighboring base pairs. Our understanding of DNA and the way it is processed in biology would profit from improved knowledge about the elementary bonds in DNA. Detailed knowledge of the time scales for breaking and forming individual base pairs and base-pair stacks would also help to make more informed decisions in the design of dynamic DNA-based nanoscale devices. RATIONALE The goal of this work is to measure the dynamics of DNA base-pair stacking at the level of individual base-pair steps. Because stacking interactions act perpendicularly to the hydrogen bonds, it should be possible to use mechanical forces to break stacking while leaving hydrogen bonds intact. To realize such measurements, we combine the positioning capabilities of DNA origami with single-molecule manipulation, as enabled by dual-beam optical traps. To make the weak single–base-pair stacking interactions experimentally accessible, we prepared parallel arrays of blunt-end DNA double helices to take advantage of avidity effects when these arrays form stacking interactions (see the figure). Our design allowed controlling the number and the sequences of the base-pair stacks. Noise-suppressing by stiff DNA origami beams connected by a flexible polymer tether enabled the repeated detection of unbinding and rebinding of stacking contacts at low forces (down to 2 piconewtons) with high time resolution (up to 1 kHz). RESULTS We sampled all 16 sequence combinations of installing a particular interfacial base pair on the array on the left beam and another base pair in the array on the right beam, and we created arrays with two, four, and six blunt ends. We could measure the force-dependent lifetimes for all base-pair step sequence combinations in the presence of 20 mM MgCl 2 , which is a condition typically used in DNA nanotechnology. For a subset of base-pair step combinations, we also obtained data in the presence of 500 mM NaCl, which mimics the conditions in the cell nucleus. The base-pair stack arrays spontaneously dissociated at average rates ranging from 0.02 to 500 per second, where the dissociation time scale strongly depended on the sequence combination and the stack array size. For a given sequence combination, larger array sizes always had larger lifetimes, as expected from avidity. Another key feature revealed in the lifetime data was the low sensitivity of the stacking interactions on the extent of pulling force. This phenomenon reflects short-ranged interaction potentials. Concerning rebinding of the stack arrays, we found that the rebinding kinetics depended much more strongly on the applied force, which may be understood by considering that rebinding of the stacks requires a thermally activated contraction of the flexible tether—which was the same for all variants—against an opposing force tether contraction. However, the rebinding kinetics was independent, within experimental error, of the base-pair step sequence combination and the size of the array under study. We used a model to estimate the free-energy increments per single base-pair stack from the kinetic rates that we measured with stack arrays. The free-energy increments per stack ranged from –0.8 to –3.4 kilocalories per mole. Our data reveals a trend in the stacking-strength hierarchy that may be associated with the extent of geometrical atomic overlap between the bases within a base-pair step. CONCLUSION Our data provides a quantitative basis for the rational design of dynamic DNA-based nanoscale machines and assemblies. Nanoengineers can directly read off the expected lifetimes of stack arrays for all sequence combinations and for various array sizes and at salt conditions that are commonly used in the field. With this data, design solutions for transition kinetics may be generated that cover several orders of magnitudes in lifetime, from milliseconds to several seconds. The sequence-resolved information obtained in our experiments may inform kinetic models of DNA hybridization and may help in adjusting force fields to perform more realistic molecular dynamics simulations. More generally, our experimental methods advance the capabilities of single-molecule mechanical experiments. Using the tethered-beam system, target molecules may be placed and exposed in controlled orientations and stoichiometry so as to study the weak forces occurring between them in solution. A variety of interactions between various kinds of molecules may be studied in the future due to the modularity and the addressability of the DNA origami–based tethered-beam system. How strong is DNA base-pair stacking? Schematic illustration of the experimental system that we developed to measure the strength of base-pair stacking on the level of single particles. The system consists of two tethered DNA origami beams that feature parallel arrays of blunt-end DNA double helices. The beams may be attached to two micrometer-sized beads for manipulation in a dual-beam optical trap. With this system, we could measure lifetimes at which DNA base-pair stacks spontaneously dissociate as a function of force applied in the helical direction.

Stimuli-responsive DNA gels that can undergo a sol-gel transition in response to photo-irradiation provide a way to engineer functional gel material with fully designed DNA base sequences. We propose an X-shaped DNA motif that turns into a gel by hybridization of self-complementary sticky ends. By embedding a photo-crosslinking artificial base in the sticky-end sequence, repetitive gel-sol transitions are achieved through UV irradiation at different wavelengths. The concentration of the DNA motif necessary for gelation is as low as 40 μm after modification of the geometrical properties of the motif. The physical properties, such as swelling degree and diffusion coefficient, were assessed experimentally.

An example of a nonspecific molecular bond is the affinity of any positive charge for any negative charge (like-unlike), or of nonpolar material for itself when in aqueous solution (like-like). This contrasts specific bonds such as the affinity of the DNA base A for T, but not for C, G, or another A. Recent experimental breakthroughs in DNA nanotechnology demonstrate that a particular nonspecific like-like bond ("blunt-end DNA stacking" that occurs between the ends of any pair of DNA double-helices) can be used to create specific "macrobonds" by careful geometric arrangement of many nonspecific blunt ends, motivating the need for sets of macrobonds that are orthogonal: two macrobonds not intended to bind should have relatively low binding strength, even when misaligned. To address this need, we introduce geometric orthogonal codes that abstractly model the engineered DNA macrobonds as two-dimensional binary codewords. While motivated by completely different applications, geometric orthogonal codes share similar features to the optical orthogonal codes studied by Chung, Salehi, and Wei. The main technical difference is the importance of 2D geometry in defining codeword orthogonality., Accepted to appear in IEEE Transactions on Molecular, Biological, and Multi-Scale Communications

Size and termini of cell-free DNA molecules circulating in blood plasma and being bound with blood cell surface of healthy females and untreated breast cancer patients were investigated. The size and concentration of circulating blood DNA were analyzed by Agilent 2100 Bioanalyser TM and TaqMan PCR. The termini of circulating DNA were examined by ligation using biotinylated double-stranded oligonucleotide adapters with random 1–3 b overhangs of both chains and subsequent quantification by PCR. Short (180 bp) and longer (>8 kbp) DNA fragments were found in cell free DNA from both groups, but short were less represented in primary breast cancer patient plasma. Predominantly high molecular weight DNA was found in cell surface bound DNA both in healthy females and breast cancer patients with a minor fraction of short fragments. Heterogeneous DNA molecules with diverse 5′- and 3′- protruding as well as blunt ends were found both in plasma DNA and cell bound DNA from healthy individuals. Cell surface bound DNA from breast cancer patients mainly contains 5′-protruding ends, whereas 5′- and 3′-protruding ends are equally presented in cell free DNA from these patients. The data obtained obviously reflect over-representation of specific nucleases in breast cancer.

The ability to design and assemble three-dimensional structures from colloidal particles is limited by the absence of specific directional bonds. As a result, complex or low-coordination structures, common in atomic and molecular systems, are rare in the colloidal domain. Here we demonstrate a general method for creating the colloidal analogues of atoms with valence: colloidal particles with chemically distinct surface patches that imitate hybridized atomic orbitals, including sp, sp2, sp3, sp3d, sp3d2 and sp3d3. Functionalized with DNA with single-stranded sticky ends, patches on different particles can form highly directional bonds through programmable, specific and reversible DNA hybridization. These features allow the particles to self-assemble into ‘colloidal molecules’ with triangular, tetrahedral and other bonding symmetries, and should also give access to a rich variety of new microstructured colloidal materials. A general method of creating colloidal particles that can self-assemble into ‘colloidal molecules’ is described: surface patches with well-defined symmetries are functionalized using DNA with single-stranded sticky ends and imitate hybridized atomic orbitals to form highly directional bonds. Chemists routinely use atoms that can form directional bonds to assemble complex and useful molecular structures. But larger colloidal particles have proved less conducive to rational assembly because they lack specific directional bonds. David Pine and colleagues now report a way around this problem that could lead to the creation of a rich variety of new micro-structured colloidal materials with technologically useful properties. Using microsphere clusters as intermediates, they create colloidal particles with chemically distinct and precisely located 'sticky patches' on the surface — up to 7 per particle — that enable specific and highly directional bonding. Using this system, they assemble 'colloidal molecules' exhibiting a wide range of bonding symmetries.

DNA is increasingly used as an important tool in programming the self-assembly of micrometer- and nanometer-scale particles. This is largely due to the highly specific thermoreversible interaction of cDNA strands, which, when placed on different particles, have been used to bind precise pairs in aggregates and crystals. However, DNA functionalized particles will only reach their true potential for particle assembly when each particle can address and bind to many different kinds of particles. Indeed, specifying all bonds can force a particular designed structure. In this paper, we present the design rules for multiflavored particles and show that a single particle, DNA functionalized with many different “flavors,” can recognize and bind specifically to many different partners. We investigate the cost of increasing the number of flavors in terms of the reduction in binding energy and melting temperature. We find that a single 2-μm colloidal particle can bind to 40 different types of particles in an easily accessible time and temperature regime. The practical limit of ∼100 is set by entropic costs for particles to align complementary pairs and, surprisingly, by the limited number of distinct “useful” DNA sequences that prohibit subunits with nonspecific binding. For our 11 base “sticky ends,” the limit is 73 distinct sequences with no unwanted overlaps of 5 bp or more. As an example of phenomena enabled by polygamous particles, we demonstrate a three-particle system that forms a fluid of isolated clusters when cooled slowly and an elastic gel network when quenched.

With the intention of investigating the DNA strand displacement properties of Plasmodium falciparum helicase PfeIF4A (formerly known as PfH45) a codon-optimized gene for expression in Escherichia coli has been produced. Several histidine-containing proteins with intrinsic helicase activity were captured from the bacterial sonicate by initial Ni 2+ -chromatography. Heparin and size-exclusion steps were subsequently required for unambiguous PfeIF4A purification. This strategy generated an active recombinant protein of significantly improved yield in comparison to previously published studies (∼4.2 mg/g wet weight of cells). Helicase unwinding assays confirmed a bipolar activity, but revealed a preference for unwinding a free 3’-end, with a rate of displacement in the 3′–5′ direction 2-fold higher than that in the 5′–3′ direction. DNA constructs with two, three or four blunt ends were not unwound. Studies confirmed the enzyme to be Mg 2+ -dependent, optimally active at 37 °C and had a background ATP turnover rate of 23.16 ± 1.74 pmol/min, which in the presence of single- or double-stranded DNA doubled to 42.92 ± 3.21 pmol/min.

Single-stranded telomeric DNA protrusions are considered to be evolutionarily conserved structural elements essential for chromosome end protection. Their formation at telomeres replicated by the leading strand mechanism is thought to involve poorly understood post-replicative processing of blunt ends. Unexpectedly, we found that angiosperm plants contain blunt-ended and short (1- to 3-nucleotide) G-overhang-containing telomeres that are stably retained in post-mitotic tissues, revealing a novel mechanism of chromosome end protection. The integrity of blunt-ended telomeres depends on the Ku70/80 heterodimer but not on another telomere capping protein, STN1. Curiously, Ku-depleted telomeres are fully functional. They are resected by exonuclease 1, promoting intrachromatid recombination, which may facilitate formation of an alternative capping structure. These data challenge the view that telomeres require ssDNA protrusions for forming a functional capping structure and demonstrate flexibility in solutions to the chromosome end protection problem.

We demonstrate the assembly of two bridged circular DNA molecules. The circles are specially engineered plasmid DNAs a few thousands base pairs in length connected by the synthetic double-stranded linker. The construct represents a very convenient system for studying DNA topology and its enzymatic transformations, since at thermodynamic equilibrium the circles have comparable probability to adopt two different topological states and the exchange between the states is an intramolecular reaction. We obtained the equilibrium distribution of these states, in which the circles form the simplest link or remain unlinked, by opening and closing the long sticky ends made in one of the bridged circles. Separation of the linked and unlinked circles by gel electrophoresis allowed us to determine the fraction of linked circles. The obtained value is in very good agreement with the results of computer simulation.

DNA is a highly effective molecule for controlling nanometer-scale structure. The convenience of using DNA lies in the programmability of Watson-Crick base-paired secondary interactions, useful both to design branched molecular motifs and to connect them through sticky-ended cohesion. Recently, the tensegrity triangle motif has been used to self-assemble three-dimensional crystals whose structures have been determined; sticky ends were reported to be the only intermolecular cohesive elements in those crystals. A recent communication in this journal suggested that tertiary interactions between phosphates and cytosine N(4) groups are responsible for intermolecular cohesion in these crystals, in addition to the secondary and covalent interactions programmed into the motif. To resolve this issue, we report experiments challenging this contention. Gel electrophoresis demonstrates that the tensegrity triangle exists in conditions where cytosine-PO(4) tertiary interactions seem ineffective. Furthermore, we have crystallized a tensegrity triangle using a junction lacking the cytosine suggested for involvement in tertiary interactions. The unit cell is isomorphous with that of a tensegrity triangle crystal reported earlier. This structure has been solved by molecular replacement and refined. The data presented here leave no doubt that the tensegrity triangle crystal structures reported earlier depend only on base pairing and covalent interactions for their formation.

A transducer consists of an input/output alphabet, a finite set of states, and a transition function. From an input symbol applied to a given state, the transition function determines the next state, and an output symbol. Using DNA, we have constructed a transducer that divides a number by 3. The input consists of a series of individually addressable 2-state DNA nanomechanical devices that control the orientations of a group of flat 6-helix DNA motifs; these motifs have edge domains tailed in sticky ends corresponding to the numbers 0 and 1. Three-domain DNA molecules (TX tiles) act as computational tiles that correspond to the transitions that the transducer can undergo. The output domain of these TX tiles contains sticky ends that also correspond to 0 or 1. Two different DNA tiles can chelate these output domains: A 5 nm gold nanoparticle is attached to the chelating tile that binds to 0-domains and a 10 nm gold nanoparticle is attached to the chelating tile that binds to 1-domains. The answer to the division is represented by the series of gold nanoparticles, which can be interpreted as a binary number. The answers of the computation are read out by examination of the transducer complexes under a transmission electron microscope. The start or end points of the output sequence can be indicated by the presence of a 15 nm gold nanoparticle. This work demonstrates two previously unreported features integrated in a single framework: [1] a system that combines DNA algorithmic self-assembly with DNA nanomechanical devices that control that input, and [2] the arrangement of non-DNA species, here metallic nanoparticles, through DNA algorithmic self-assembly. The nanomechanical devices are controlled by single-stranded DNA strands, allowing multiple input sequences to be applied to the rest of the system, thus guiding the algorithmic self-assembly to a variety of outputs.

A computability model based on DNA sequences called sticker systems was introduced by Kari et al. (1998). Sticker systems are language generating devices based on the sticker operation. In this work, a theoretical study about a new class of P system based on the sticker operation has been proposed. In this system, objects are double stranded sequences with sticky ends and evolution rules are sticking rules based on sticker operation. The authors compare the language generated by the proposed system with regular languages. The authors suggest another P system based on the bidirectional sticker system.

During DNA replication by the λ-like bacteriophages, immature concatemeric DNA is produced by rolling circle replication. The concatemers are processed into mature chromosomes with cohesive ends, and packaged into prohead shells, during virion assembly. Cohesive ends are generated by the viral enzyme terminase, which introduces staggered nicks at cos, an approx. 200 bp-long sequence containing subsites cosQ, cosN and cosB. Interactions of cos subsites of immature concatemeric DNA with terminase orchestrate DNA processing and packaging. To initiate DNA packaging, terminase interacts with cosB and nicks cosN. The cohesive ends of N15 DNA differ from those of λ at 2/12 positions. Genetic experiments show that phages with chromosomes containing mismatched cohesive ends are functional. In at least some infections, the cohesive end mismatch persists through cyclization and replication, so that progeny phages of both allelic types are produced in the infected cell. N15 possesses an asymmetric packaging specificity: N15 DNA is not packaged by phages λ or 21, but surprisingly, N15-specific terminase packages λ DNA. Implications for genetic interactions among λ-like bacteriophages are discussed.

Perylene bisimides (PBI) have been synthetically incorporated as caps onto a Y-shaped DNA triple strand. These PBI caps serve as "sticky" ends in the spontaneous assembly of larger DNA ensembles, linking the triangular DNA through stacking interactions. This, in turn, yields a hypsochromic shift in the absorption and a red shift in the fluorescence as characteristic optical readouts. This assembly occurs spontaneously without any enzymatic ligation process and without the use of overhanging DNA as sticky ends. Instead, dimerizations of the PBI chromophores in the assembly are controlled by the DNA as a structural scaffold. Thereby, the PBI-driven assembly is fully reversible. Due to the fact that PBI dimerization does not occur in the single strand, the aggregates can be destroyed by thermal dehybridization of the DNA scaffold and reassembled by reannealing of the DNA construct. In view of the fact that PBI forms stable radical anions, the presented DNA architectures are not only interesting optical biomaterials, but are also promising materials for molecular electronics with DNA.

We report the formation of liquid crystalline (LC) phases of short double-stranded DNA with non-pairing (non-sticky) overhangs, confined between two-dimensional (2D) lipid bilayers of cationic liposome–DNA complexes. In a landmark study (Science 2007, 318, 1276), Nakata et al. reported on the discovery of strong end-to-end stacking interactions between short DNAs (sDNAs) with blunt ends leading to the formation of 3D nematic (N) and columnar LC phases. Employing synchrotron small-angle X-ray scattering, we have studied the interplay between shape anisotropy-induced and DNA end-to-end interaction-induced N ordering for 11 bp, 24 bp, and 48 bp sDNA rods with single-stranded oligo-thymine (T) overhangs modulating the end-to-end interactions. For suppressed stacking interactions with 10-T overhangs, the volume fraction of sDNA at which the 2D isotropic (I) to N transition occurs for 24 bp and 48 bp sDNA rods depended on their length to width (L/D) shape anisotropy, qualitatively consistent with Onsager's theory for the entropic alignment of rigid rods. As the overhang length is reduced from 10 T to 5 T and 2 T for 24 bp and 48 bp sDNA, the N to I transition occurs at lower volume fractions, indicating the onset of some degree of end-to-end stacking interactions. The 11 bp sDNA rods with 5-T and 10-T overhangs remain in the I phase, consistent with their small shape anisotropy (L/D ≈ 1.9) below the limit for Onsager LC ordering. Unexpectedly, in contrast to the behavior of 24 bp and 48 bp sDNA, the end-to-end interactions between 11 bp sDNA rods with 2-T overhangs set in dramatically and a novel 2D columnar N phase (NC) with finite-length columns formed. The building blocks of this phase are comprised of 1D stacks of (on average) four 11bp DNA-2T rods with an effective LSTACKED/D ≈ 8.2. Our findings have implications for the DNA-directed assembly of nanoparticles on 2D platforms via end-to-end interactions and in designing optimally packed LC phases of short anisotropic biomolecules (such as peptides and short-interfering RNAs) on nanoparticle membranes, which are used in gene silencing and chemical delivery.

Microtubules (MTs) exhibit dynamic instability, alternating between phases of growth and shortening, mostly at their uncapped plus ends. Based on results from cryo-electron microscopy it was proposed that growing MTs display mainly curved sheets and blunt ends; during depolymerisation curled ‘ramshorns’ predominate. Observations of MTs in mitotic cells have suggested that the situation in vivo differs from that in vitro, but so far, a clear comparison between in vivo and in vitro results has not been possible because MT end structures could not be correlated directly with the dynamic state of that particular MT. Here we combine light microscopy and electron tomography (ET) to show that growing MT plus ends in the fission yeast Schizosaccharomyces pombe display predominantly a flared morphology. This indicates that MT polymerisation in vivo and in vitro can follow different paths.

Virulent phages of the Siphoviridae family are responsible for milk fermentation failures worldwide. Here, we report the characterization of the product of the early expressed gene orf35 from Lactococcus lactis phage p2 (936 group). ORF35(p2), also named Sak3, is involved in the sensitivity of phage p2 to the antiviral abortive infection mechanism AbiK. The localization of its gene upstream of a gene coding for a single-strand binding protein as well as its membership to a superfamily of single-strand annealing proteins (SSAPs) suggested a possible role in homologous recombination. Electron microscopy showed that purified ORF35(p2) form a hexameric ring-like structure that is often found in proteins with a conserved RecA nucleotide-binding core. Gel shift assays and surface plasmon resonance data demonstrated that ORF35(p2) interacts preferentially with single-stranded DNA with nanomolar affinity. Atomic force microscopy showed also that it preferentially binds to sticky DNA substrates over blunt ends. In addition, in vitro assays demonstrated that ORF35(p2) is able to anneal complementary strands. Sak3 also stimulates Escherichia coli RecA-mediated homologous recombination. Remarkably, Sak3 was shown to possess an ATPase activity that is required for RecA stimulation. Collectively, our results demonstrate that ORF35(p2) is a novel SSAP stimulating homologous recombination.

Terms to be familiar with before you start to solve the test: 3'- and 5'-ends of DNA fragments, plasmids, restriction endonucleases, agarose gel electrophoresis, linear and circular DNA, superhelical DNA, blunt and sticky ends, DNA ligase, nuclease, [γ-(32) P] ATP, and radioactive labeling. © 2014 by The International Union of Biochemistry and Molecular Biology, 42(4):348-350, 2014.

Nanotechnology aims to organize matter with the highest possible accuracy and control. Such control will lead to nanoelectronics, nanorobotics, programmable chemical synthesis, scaffolded crystals, and nanoscale systems responsive to their environments. Structural DNA nanotechnology1 is one of the most powerful routes to this goal. It combines robust branched DNA species with the control of affinity and structure2 inherent in the programmability of sticky ends. The successes of structural DNA nanotechnology include the formation of objects,3 2D crystals,4 3D crystals,5 nanomechanical devices,6 and various combinations of these species (e.g., ref. 7). DNA origami8 is arguably the most effective way of producing a large addressable area on a 2D DNA surface. This method entails the combination of a long single strand (typically M13 single-stranded form, 7249 nucleotides) with about ~250 staple strands to define its shape and patterning. With a pixilation estimated at about 6 nm,8 it is possible to build patterns with about 100 addressable points within a definable shape in an area of about 10,000 nm2. Many investigators have sought unsuccessfully to increase the useful size of 2D origami units by forming crystals of individual origami tiles.0 Here, we report the 2D crystallization of origami tiles to yield a 2D array with dimensions 2–3 microns on an edge. This size is likely to be large enough to connect bottom-up methods of patterning with top-down approaches.

The PD-(D/E)XK type II restriction endonuclease ThaI cuts the target sequence CG/CG with blunt ends. Here, we report the 1.3 Å resolution structure of the enzyme in complex with substrate DNA and a sodium or calcium ion taking the place of a catalytic magnesium ion. The structure identifies Glu54, Asp82 and Lys93 as the active site residues. This agrees with earlier bioinformatic predictions and implies that the PD and (D/E)XK motifs in the sequence are incidental. DNA recognition is very unusual: the two Met47 residues of the ThaI dimer intercalate symmetrically into the CG steps of the target sequence. They approach the DNA from the minor groove side and penetrate the base stack entirely. The DNA accommodates the intercalating residues without nucleotide flipping by a doubling of the CG step rise to twice its usual value, which is accompanied by drastic unwinding. Displacement of the Met47 side chains from the base pair midlines toward the downstream CG steps leads to large and compensating tilts of the first and second CG steps. DNA intercalation by ThaI is unlike intercalation by HincII, HinP1I or proteins that bend or repair DNA.

The MvaI restriction endonuclease cuts 5'-CC↓AGG-3'/5'-CC↑TGG-3' sites as indicated by the arrows. N4-methylation of the inner cytosines (C(m4)CAGG/C(m4)CTGG) protects the site against MvaI cleavage. Here, we show that MvaI nicks the G-strand of the related sequence (CCGGG/CCCGG, BcnI site) if the inner cytosines are C5-methylated: C(m5)C↓GGG/CC(m5)CGG. At M.SssI-methylated SmaI sites, where two oppositely oriented methylated BcnI sites partially overlap, double-nicking leads to double-strand cleavage (CC(m5)C↓GGG/CC(m5)C↑GGG) generating fragments with blunt ends. The double-strand cleavage rate and the stringency of substrate site recognition is lower at the methylation-dependent site than at the canonical target site. MvaI is the first restriction endonuclease shown to possess, besides the 'normal' activity on its unmethylated recognition site, also a methylation-directed activity on a different sequence.

In this paper, a simple universal high-throughput method of constructing the vectors was developed. It could add proper adapter when the PCR primers were designed, and the purpose fragments were cloned by PCR, and the various complementary sticky ends were created by T4 DNA polymerase's 3' -exodeoxyribonuclease activity. If all these fragments were put together with DNA ligase, they would recombinate in an orientation. If they had been transformated, the tansformants would be identified. Let's take the Oryza sativa single-cross homologous recombination chloroplast expression vector pRSMGA which was constructed with seven fragments as an example, if the vector pRSMGA was constructed in using the method what had mentioned, only twice recombination and transformation would be done. Scores of experiments had proved that it is a simple universal high-throughput novel method to construct the complicated vectors, which has not appeared in the periodical.

A simplified origami strategy to create large-scale complex DNA nanostructures based on the rolling circle amplification (RCA) is developed. The long repetitive block single strand DNA (ssDNA) synthesized from RCA with a few staple strands are used to fabricate DNA origami, which can be assembled into the first level assemblies of micro-scale length mono-nanoladders. Depending on base pairing among the sticky ends of short staple strands in nanoladders, the second level assemblies with large-scale and controlled structures such as tri-nanoladder, penta-nanoladder, and even nanobrocade, can be further achieved.

Peptides comprised entirely of β3-amino acids, commonly referred to as β-foldamers, have been shown to self-assemble into a range of materials. Previously, β-foldamers have been functionalised via various side chain chemistries to introduce function to these materials without perturbation of the self-assembly motif. Here, we show that insertion of both rigid and flexible molecules into the backbone structure of the β-foldamer did not disturb the self-assembly, provided that the molecule is positioned between two β3-tripeptides. These hybrid β3-peptide flanked molecules self-assembled into a range of structures. α-Arginlyglycylaspartic acid (RGD), a commonly used cell attachment motif derived from fibronectin in the extracellular matrix, was incorporated into the peptide sequence in order to form a biomimetic scaffold that would support neuronal cell growth. The RGD-containing sequence formed the desired mesh-like scaffold but did not encourage neuronal growth, possibly due to over-stimulation with RGD. Mixing the RGD peptide with a β-foldamer without the RGD sequence produced a well-defined scaffold that successfully encouraged the growth of neurons and enabled neuronal electrical functionality. These results indicate that β3-tripeptides can form distinct self-assembly units separated by a linker and can form fibrous assemblies. The linkers within the peptide sequence can be composed of a bioactive α-peptide and tuned to provide a biocompatible scaffold.

DNA ligases are key enzymes in molecular and synthetic biology that catalyze the joining of breaks in duplex DNA and the end-joining of DNA fragments. Ligation fidelity (discrimination against the ligation of substrates containing mismatched base pairs) and bias (preferential ligation of particular sequences over others) have been well-studied in the context of nick ligation. However, almost no data exist for fidelity and bias in end-joining ligation contexts. In this study, we applied Pacific Biosciences Single-Molecule Real-Time sequencing technology to directly sequence the products of a highly multiplexed ligation reaction. This method has been used to profile the ligation of all three-base 5′-overhangs by T4 DNA ligase under typical ligation conditions in a single experiment. We report the relative frequency of all ligation products with or without mismatches, the position-dependent frequency of each mismatch, and the surprising observation that 5′-TNA overhangs ligate extremely inefficiently compared to all other Watson–Crick pairings. The method can easily be extended to profile other ligases, end-types (e.g. blunt ends and overhangs of different lengths), and the effect of adjacent sequence on the ligation results. Further, the method has the potential to provide new insights into the thermodynamics of annealing and the kinetics of end-joining reactions.

In recent years, DNA self-assembly is beginning to be seen as a practical vehicle for computation. A number of research groups have begun developing nanofabrication methods based on DNA self-assembly. Since the basic logical operations based on DNA molecules could be considered as the fundamental units of DNA computer architecture, how to achieve the basic logical operations based DNA self-assembly is explored in this paper. Original intention on tiling can be applied to the assembly and realization of circuits. These procedures can be realized on the molecular scale through the medium of self-assembled DNA tiles. One layer of self-assembled DNA tiles will be used as self-assembly the program or circuit that leads to the computation of a basic Boolean expression. For this purpose, a modified triple-crossover (TX) tiles which consist of two inputs and one output are used to execute molecular logical operations. The self-assembly of designed periodic one-dimensional arrays are composed of modified triple-crossover (TX) tiles, in which the intermolecular contacts are directed by sticky ends. Here, the paper makes a further to perform a one-dimensional algorithmic self-assembly of DNA triple-crossover molecules that can be used to execute five steps of a logical (cumulative AND and OR) operations on a string of binary bits.

MutS is a DNA repair protein that recognizes unpaired and bulged bases. When it binds to DNA, it bends the double helix. We have developed a novel DNA-based nanomechanical device that measures the amount of work that a DNA-bending protein can do when it binds to the double helix. The device we report here is a scissors-like device consisting of two double-crossover (DX) molecules connected to each other by a flexible Holliday junction. The two DX components are connected by a double helix that contains the binding site for MutS; when the binding site duplex is bent, the scissors contracts. The two DX molecules are also joined by sticky ends on an edge adjacent to the binding site; the sticky ends can be disrupted if the protein binds with sufficient free energy. Those sticky ends are flanked by a pair of dyes; when the sticky ends are disrupted, the dyes separate, and the fluorescence resonance energy transfer signal can monitor the disruption. The strength of the sticky ends is readily varied, so that the ability of the protein to disrupt them can be quantitated. We use this device to measure work in conjunction with a second device that measures the bending angle resulting from protein binding, so as to calibrate the system. Our data are in good agreement with previous measurements of MutS binding, indicating that this device is able to measure the strength of binding correctly.

We continue the formal study of multiset-based self-assembly. The process of self-assembly of graphs, where iteratively new nodes are attached to a given graph, is guided by rules operating on nodes labelled by multisets. In this way, the multisets and rules model connection points (such as "sticky ends") and complementarity/affinity between connection points, respectively. We identify three natural ways (individual, free, and collective) to attach (aggregate) new nodes to the graph, and study the generative power of the corresponding self-assembly systems. For example, it turns out that individual aggregation can be simulated by free or collective aggregation. However, we demonstrate that, for a fixed set of connection points, collective aggregation is rather restrictive. We also give a number of results that are independent of the way that aggregation is performed.

Tyrosyl-DNA-phosphodiesterase 1 (Tdp1) can disjoin peptides covalently bound to DNA. We assessed the role of Tdp1 in nonhomologous end joining (NHEJ) and found that linear DNA molecules with 5′ extensions showed a high frequency of misrepair in Δ tdp 1 cells. The joining errors in Δ tdp 1 cells were predominantly 2-4 nucleotide insertions. Ends with 3′ extensions or blunt ends did not show enhanced frequencies of errors, although Δ tdp 1 cells repaired blunt DNA ends with greater efficiency than WT cells. We found that insertions required Ku80 and DNA ligase IV, as well as polymerase IV. Our results show that yeast Tdp1 is a component of the NHEJ pathway. We suggest that Tdp1p 3′ nucleosidase activity regulates the processing of DNA ends by generating a 3′ phosphate, thereby restricting the ability of polymerases and other enzymes from acting at DNA ends. In support of this model, we found that overexpression of Tpp1, a yeast DNA 3′ phosphatase, also leads to a higher frequency of insertions, suggesting that the generation of a 3′ phosphate is a key step in Tdp1-mediated error prevention during NHEJ.

This study focused on concatemer formation and integration pattern of transgenes in zebrafish embryos. A reporter plasmid based on enhanced green fluorescent protein (eGFP) driven by Cytomegalovirus (CMV) promoter, pCMV-pax6in-eGFP, was constructed to reflect transgene behavior in the host environment. After removal of the insertion fragment by double digestion with various combinations of restriction enzymes, linearized pCMV-pax6in-eGFP vectors were generated with different combinations of 5'-protruding, 3'-protruding, and blunt ends that were microinjected into zebrafish embryos. Repair of double-strand breaks (DSBs) was monitored by GFP expression following religation of the reporter gene. One-hundred-and-ninety-seven DNA fragments were amplified from GFP-positive embryos and sequenced to analyze the repair characteristics of different DSB end combinations. DSBs involving blunt and asymmetric protruding ends were repaired efficiently by direct ligation of blunt ends, ligation after blunting and fill-in, or re moved by cutting. Repair of DSBs with symmetric 3'-3' protrusions was less efficient and utilized template-directed repair. The results suggest that non-homologous end joining (NHEJ) was the principal mechanism of exogenous gene concatemer formation and integration of transgenes into the genome of transgenic zebrafish.

We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA.