Sticky Ends Research Articles

Enzymatic Assembly of DNA Nanostructures and Fragments with Sequence Overlaps.

Homologous recombination, an evolutionarily conserved DNA double-strand break repair pathway to protect genome stability, has long been exploited for the in vivo and in vitro assembly of multiple DNA duplex fragments in molecular cloning. Whether such methods can also be applied in the self-assembly of DNA nanostructures remains underexplored. Here, we report an enzymatic approach for the self-assembly of high-order DNA constructs with overlapping segments. In our system, a DNA polymerase with exonuclease activity was introduced to produce ssDNA overhangs for specific sticky end cohesion, and as many as 25 DNA structural units were designed to be hierarchically assembled. Using this approach, we successfully constructed a variety of high-order DNA nanostructures, including tubes and extended oligomers, from homogeneous assembly and custom multimers from heterogeneous assembly. Our strategy expands the construction toolbox of complex DNA nanostructures and highlights the potential to enhance the assembly of duplex fragments in molecular cloning.

Novel bispecific aptamer targeting PD-1 and nucleolin for cancer immunotherapy

BackgroundImmune checkpoint blockade (ICB) is a promising strategy for cancer treatment and has achieved remarkable clinical results. Further improvement of ICB efficacy may advance cancer immunotherapy and has evident medical importance. Here in this study, a PD-1 aptamer was functionalized with a tumor-homing nucleolin aptamer (AS1411) to build a novel bispecific agent (BiApt) for boosting the efficacy of ICB therapy.ResultsThe two aptamers were coupled together via sticky ends to form BiApt, which had an average size of 11.70 nm. Flow cytometry revealed that BiApt could bind with both the activated T cells and the nucleolin-expressing tumor cells. In addition, BiApt could recruit more T cells to the vicinity of nucleolin-positive tumor cells. Functionally, BiApt enhanced the PBMC-mediated anticancer cytotoxicity in vitro compared with free PD-1 aptamer. Moreover, in an animal model of CT26 colon cancer, BiApt significantly boosted the antitumor efficacy vs. free PD-1 aptamer.ConclusionThe results suggest that bispecific agent combining ICB and tumor-homing functions has potential to improve the efficacy of ICB immunotherapy.

A high sensitivity strategy of DNMT1 activity detection based on self-assembled nucleic acid probe signal amplification technique

The abnormal expression of DNA methyltransferase1 (DNMT1) leads to change of genome methylation pattern, which may result in the occurrence and development of cancer. Therefore, the determination of DNMT activity is of great significance for cancer diagnosis and drug screening. In this study, we developed a novel method to detect DNMT1 activity using self-assembly nucleic acid probe signal amplification based on a fluorescence assay. The semi-methylated Biotin-S1'-S2' with sticky ends was fixed to the magnetic beads through the affinity of streptavidin and Biotin. Afterward, pre-prepared poly Tetramethylrhodamine (TAMRA) was added for base complementary pairing with Biotin-S1'-S2' via its sticky end when DNMT1 is present and BssHII shearing is blocked by fully methylated bilayers, thus, the amplified fluorescent signal can be detected. The results showed that the fluorescence intensity of the system was positively correlated with the concentration of DNMT1 in the concentration range of 1–100 nmol/L, and the detection limit was as low as 0.5 nmol/L. The method is simple, highly visualized and successfully applied for the recovery of DNMT1 activity in serum samples. Thus, the method shows great potential for application in clinical diagnosis related to DNMT1.

Target-catalyzed self-assembled spherical G-quadruplex/hemin DNAzymes for highly sensitive colorimetric detection of microRNA in serum.

The accurate and visual detection of circulating microRNA (miRNA) has attracted increasing interest due to its pivotal role in clinical disease diagnosis. Taking advantages of nucleic acid isothermal amplification and enzyme-catalyzed chromogenic reaction, here, a colorimetric sensing strategy was proposed for sensitive miRNA analysis. When the target miRNA was present, local catalytic hairpin assembly (CHA) would be triggered and proceed continuously to form dozens of double-stranded oligonucleotides with G-rich sticky ends on the gold nanoparticle, which could self-assemble into a spherical G-quadruplex (GQ)/hemin DNAzyme by binding with hemin and potassium ions. As a horseradish peroxidase-mimic, GQ/hemin DNAzyme could catalyze the redox reaction and color change of the substrates. Taking miRNA-21 as an example, the developed method exhibited satisfactory specificity as well as high sensitivity with a detection limit of 90.3fM. Furthermore, the sensing platform has been successfully employed to detect miRNA-21 in spiked serum, providing a promising tool for early diagnosis of cancers.

Understanding the self-assembly dynamics of A/T absent 'four-way DNA junctions with sticky ends' at altered physiological conditions through molecular dynamics simulations.

Elucidation of structure and dynamics of alternative higher-order structures of DNA such as in branched form could be targeted for therapeutics designing. Herein, we are reporting the intrinsically dynamic and folds transitions of an unusual DNA junction with sequence d(CGGCGGCCGC)4 which self-assembles into a four-way DNA junction form with sticky ends using long interval molecular simulations under various artificial physiological conditions. The original crystal structure coordinates (PDB ID: 3Q5C) for the selected DNA junction was considered for a total of 1.1 μs molecular dynamics simulation interval, including different temperature and pH, under OPLS-2005 force field using DESMOND suite. Following, post-dynamics structure parameters for the DNA junction were calculated and analyzed by comparison to the crystal structure. We show here that the self-assembly dynamics of DNA junction is mitigated by the temperature and pH sensitivities, and discloses peculiar structural properties as function of time. From this study it can be concluded on account of temperature sensitive and pH dependent behaviours, DNA junction periodic arrangements can willingly be synthesized and redeveloped for multiple uses like genetic biomarkers, DNA biosensor, DNA nanotechnology, DNA Zipper, etc. Furthermore, the pH dis-regulation behaviour may be used to trigger the functionality of DNA made drug-releasing nanomachines.

Programmable 3D Hexagonal Geometry of DNA Tensegrity Triangles

AbstractNon‐canonical interactions in DNA remain under‐explored in DNA nanotechnology. Recently, many structures with non‐canonical motifs have been discovered, notably a hexagonal arrangement of typically rhombohedral DNA tensegrity triangles that forms through non‐canonical sticky end interactions. Here, we find a series of mechanisms to program a hexagonal arrangement using: the sticky end sequence; triangle edge torsional stress; and crystallization condition. We showcase cross‐talking between Watson–Crick and non‐canonical sticky ends in which the ratio between the two dictates segregation by crystal forms or combination into composite crystals. Finally, we develop a method for reconfiguring the long‐range geometry of formed crystals from rhombohedral to hexagonal and vice versa. These data demonstrate fine control over non‐canonical motifs and their topological self‐assembly. This will vastly increase the programmability, functionality, and versatility of rationally designed DNA constructs.

Programmable 3D Hexagonal Geometry of DNA Tensegrity Triangles.

Non-canonical interactions in DNA remain under-explored in DNA nanotechnology. Recently, many structures with non-canonical motifs have been discovered, notably a hexagonal arrangement of typically rhombohedral DNA tensegrity triangles that forms through non-canonical sticky end interactions. Here, we find a series of mechanisms to program a hexagonal arrangement using: the sticky end sequence; triangle edge torsional stress; and crystallization condition. We showcase cross-talking between Watson-Crick and non-canonical sticky ends in which the ratio between the two dictates segregation by crystal forms or combination into composite crystals. Finally, we develop a method for reconfiguring the long-range geometry of formed crystals from rhombohedral to hexagonal and vice versa. These data demonstrate fine control over non-canonical motifs and their topological self-assembly. This will vastly increase the programmability, functionality, and versatility of rationally designed DNA constructs.

Symmetry-Breaking Dendrimer Synthons in Colloidal Crystal Engineering with DNA

Breaking symmetry in colloidal crystals is challenging due to the inherent chemical and structural isotropy of many nanoscale building blocks. If a non-particle component could be used to anisotropically encode such building blocks with orthogonal recognition properties, one could expand the scope of structural and compositional possibilities of colloidal crystals beyond what is thus far possible with purely particle-based systems. Herein, we report the synthesis and characterization of novel DNA dendrimers that function as symmetry-breaking synthons, capable of programming anisotropic and orthogonal interactions within colloidal crystals. When the DNA dendrimers have identical sticky ends, they hybridize with DNA-functionalized nanoparticles to yield three distinct colloidal crystals, dictated by dendrimer size, including a structure not previously reported in the field of colloidal crystal engineering, Si2Sr. When used as symmetry-breaking synthons (when the sticky ends deliberately consist of orthogonal sequences), the synthesis of binary and ternary colloidal alloys with structures that can only be realized through directional interactions is possible. Furthermore, by modulating the extent of shape anisotropy within the DNA dendrimers, the local distribution of the nanoparticles within the crystals can be directed.

Network Phases with Multiple-Junction Geometries at the Gyroid-Diamond Transition.

A novel phase sequence for the transition from the double diamond to the double gyroid cubic phases via two non-cubic intermediate phases, an orthorhombic Fmmm (O69) phase and a hexagonal P63/m (H176) phase, is reported for specifically designed bolapolyphiles composed of a linear rod-like bistolane core with sticky glycerol ends and two branched central and two linear peripheral side chains. These liquid crystalline (LC) phases represent members of a new class of unicontinuous network phases, formed by longitudinal rod bundles with polar spheres acting as junctions and the alkyl chains forming the continuum around them. In contrast to previously known bicontinuous cubic networks, they combine different junctions with different angles in a common structure, and one of them even represents a triple network instead of the usually found double networks. This provides new perspectives for the design of soft network phases with enhanced structural complexity, inspiring the search for new supramolecular networks, nano-particle arrays, and photonic band-gap materials.

Effects of solvent conditions on the self-assembly of heterotrimeric collagen-like peptide (CLP) triple helices: a coarse-grained simulation study.

We perform coarse-grained (CG) molecular dynamics (MD) simulations to investigate the self-assembly of collagen-like peptide (CLP) triple helices into fibrillar structures and percolated networks as a function of solvent quality. The focus of this study is on CLP triple helices whose strands are different lengths (i.e., heterotrimers), leading to dangling 'sticky ends'. These 'sticky ends' are segments of the CLP strands that have unbonded hydrogen-bonding donor/acceptor sites that drive heterotrimeric CLP triple helices to physically associate with one another, leading to assembly into higher-order structures. We use a validated CG model for CLP in implicit solvent and capture varying solvent quality through changing strength of attraction between CG beads representing the amino acids in the CLP strands. Our CG MD simulations show that, at lower CLP concentrations, CLP heterotrimers assemble into fibrils and, at higher CLP concentrations, into percolated networks. At higher concentrations, decreasing solvent quality causes (i) the formation of heterogeneous network structures with a lower degree of branching at network junctions and (ii) increases in the diameter of network strands and pore sizes. We also observe a nonmonotonic effect of solvent quality on distances between network junctions due to the balance between heterotrimer end-end associations driven by hydrogen bonding and side-side associations driven by worsening solvent quality. Below the percolation threshold, we observe that decreasing solvent quality leads to the formation of fibrils composed of multiple aligned CLP triple helices, while the number of 'sticky ends' governs the spatial extent (radius of gyration) of the assembled fibrils.

RNA-DNA hybrid nano-materials for highly efficient and long lasting RNA interference effect.

In attempts to effectively improve RNAi function, we herein report a new RNAi approach using X-shaped RDNA and Dgel (RNA interfering DNA hydrogel, Ri-Dgel). X-shaped RDNA is a 4 branched nanostructure which was composed of three dsDNA branches and one dsRNA branch, and the structure was made by annealing partially complementary ssDNAs and chimeric RNA-DNA oligonucleotides. Ri-Dgel was synthesized through the ligation of the X-shaped RDNAs using their palindromic sticky ends. In MDCK/GFP cells transfected with 1 μM of each format of siRNA, Ri-Dgel and X-RDNA, the intensity of GFP fluorescence was significantly reduced by 65% and 56%, respectively, while dsRNA which is a conventional siRNA format showed a relatively weak reduction intensity of 7% compared with a negative control. We also observed the decreased intensity of GFP fluorescence by approximately 59% in MDA-MB-231/GFP cells transfected with 5 nM Ri-Dgel. Furthermore, the Ri-Dgel showed persistent RNAi efficiency up to 6 days from the treatment. The use of Ri-Dgel to trigger RNAi resulted in enhanced efficacy and longer duration at lower concentration compared to traditional dsRNA implying the nanostructured DNA-RNA hybrid materials have great potential as a platform technology for RNAi-based therapy.

Sequence-dependent fusion dynamics and physical properties of DNA droplets.

Liquid-liquid phase separation (LLPS) of biopolymer molecules generates liquid-like droplets. Physical properties such as viscosity and surface tension play important roles in the functions of these droplets. DNA-nanostructure-based LLPS systems provide useful model tools to investigate the influence of molecular design on the physical properties of the droplets, which has so far remained unclear. Herein, we report changes in the physical properties of DNA droplets by sticky end (SE) design in DNA nanostructures. We used a Y-shaped DNA nanostructure (Y-motif) with three SEs as a model structure. Seven different SE designs were used. The experiments were performed at the phase transition temperature where the Y-motifs self-assembled into droplets. We found that the DNA droplets assembled from the Y-motifs with longer SEs exhibited a longer coalescence period. In addition, the Y-motifs with the same length but different sequence SEs showed slight variations in the coalescence period. Our results suggest that the SE length greatly affected the surface tension at the phase transition temperature. We believe that these findings will accelerate our understanding of the relationship between molecular design and the physical properties of droplets formed via LLPS.

Geometry guided crystallization of anisotropic DNA origami shapes.

Three-dimensional assembly based on DNA origami structures is an ideal method to precisely fabricate nano-scale materials. Additionally, applying an anisotropic assembly unit facilitates constructing complex materials with extraordinary structure. However, it still remains challenging to crystallize anisotropic DNA nano-structures using simple design, because the assembly of low-symmetry monomers often requires harsh auxiliary conditions and more complicated crystallization processes. In this work, we managed to crystallize the anisotropic elongated octahedral DNA origami frames by non-specific connections, and acquired two kinds of highly ordered superlattices purely by conducting multiple annealing processes and increasing the rigidity of the connection parts. In the case where the connection parts were composed of soft DNA sticky ends, we obtained the theoretically inaccessible simple cubic superlattices by this anisotropic DNA origami shape. Through characterization by small-angle X-ray scattering and scanning electron microscopy, we found that the DNA monomers are arbitrarily arranged due to the stress buffering of the soft DNA SEs, while in the stiffer case, simple tetragonal superlattices with translational arrangement of most anisotropic DNA origami shapes were synthesized as expected. This work deepened the understanding of geometry-guided crystallization of DNA origami shapes and provided a new path for constructing three-dimensional functional devices with simple design.

Sensitive and convenient detection of miRNA-145 using a gold nanoparticle-HCR coupled system: computational and in vitro validations.

Multiple sclerosis (MS) remains a challenging disease that requires timely diagnosis. Therefore, an ultrasensitive optical biosensor based on hybridization chain reaction (HCR) was developed to detect microRNA-145 (miRNA-145) as an MS biomarker. To construct such a sensor, HCR occurred between specific hairpin probes, as MB1 contains a poly-cytosine nucleotide loop and MB2 has a poly-guanine nucleotide sticky end. By introducing miR-145 as a target sequence, long-range dsDNA polymers are formed. Then, positively charged gold nanoparticles (AuNPs) were incubated with the HCR product, which adsorbed onto the dsDNA polymers due to electrostatic adsorption. This resulted in the precipitation of the AuNPs. By incubating different concentrations of miR-145 with AuNPs, the changes in the UV-vis spectrum of the supernatant were analyzed. The proposed biosensor showed a great ability to detect miR-145 in a wide linear range from 1 pM-1 nM with an excellent detection limit (LOD) of 0.519 nM. Furthermore, the developed biosensor indicated considerable selectivity in discriminating between miR-145 and mismatched sequences. It shows high selectivity in differentiating targets. Interestingly, the proposed method was also able to detect miRNA-145 in the diluted serum samples. In conclusion, this sensing platform exhibits high selectivity and specificity for the detection of circulating microRNAs, which holds great promise for translation to routine clinical applications.

Stimulus-responsive and dual-target DNA nanodrugs for rheumatoid arthritis treatment

Tumor necrosis factor receptor-1 (TNFR1) and DEK are closely associated with the development of rheumatoid arthritis (RA). Taking advantage of the high adenosine triphosphate (ATP) in RA microenvironment and the interactions of DNA aptamers with their targets, an ATP-responsive DNA nanodrug was constructed that simultaneously targets TNFR1 and DEK for RA therapy. To this end, DEK target aptamer DTA and TNFR1 target aptamer Apt1-67 were equipped with sticky ends to hybridize with ATP aptamer (AptATP) and fabricated DNA nanodrug DAT. Our results showed that DAT was successfully prepared with good stability. In the presence of ATP, DAT was disassembled, resulting in the release of DTA and Apt1-67. In vitro studies demonstrated that DAT was superior to the non-responsive DNA nanodrug TD-3A3T in terms of anti-inflammation activity and ATP was inevitable to maximize the anti-inflammation ability of DAT. The superior efficacy of DAT is attributed to the more potent inhibition of caspase-3 and NETs formation. In vivo results further confirmed the anti-RA efficacy of DAT, whereas the administration routes (intravenous injection and transdermal administration via microneedles) did not cause significant differences. Overall, the present study supplies an intelligent strategy for RA therapy and explores a promising administration route for future clinical medication of RA patients.

Ferrocene-Functionalized Covalent Organic Frameworks and Target Catalyzed Hairpin Assembly Strategy for Amplified Electrochemical Determination of MicroRNAs.

MicroRNAs (miRNAs) are considered as significant biomarkers in early diagnosis and treatment of diseases. Herein, an electrochemical biosensor that uses ferrocene (Fc)-functionalized covalent organic frameworks (COFs), a DNA tetrahedron nanostructure (DTN) biosensing interface, and a target catalyzed hairpin assembly (CHA) strategy has been fabricated and successfully developed for the sensitive and specific determination of microRNA-21 (miR-21). The COF served as a linked substrate for immobilization of gold nanoparticles (AuNPs), Fc-COOH, and complementary DNA probe L1 to prepare the electrochemical signal probe COF/Au/Fc/L1, which has a large surface area, extraordinary catalytic properties, and superior biocompatibility to amplify the current signal. The DTN containing a hairpin sequence H1 at one vertex was elaborately designed to construct the biosensing interface; thus, the CHA could be implemented on the electrode surface. In the presence of miR-21, the CHA reaction between H1 and the hairpin H2 was triggered to produce a great number of duplex DNA (H1/H2) with sticky ends. Then, the signal probe COF/Au/Fc/L1 was modified on the electrode surface through the hybridization between L1 and the sticky end of H1/H2, thereby obtaining an amplified Fc current signal. Under optimal conditions, the biosensor showed a wide linear response ranging from 1 fM to 10 nM miR-21, with a low detection limit of 0.33 fM (S/N = 3). Meanwhile, the method showed acceptable accuracy and precision for the determination of miR-21 in human serum.

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.

Detection of apoptotic cells based on in situ hybridization chain reaction using specific hairpins.

There are an increasing number of experiments to study programmed cell death/apoptosis, one of the characteristics of which is DNA fragmentation. The only current method for in situ detection of DNA fragmentation is Terminal deoxynucleotidyl transferase mediated-dUTP Nick End Labeling, TUNEL. In this study, a new method for in situ detection of apoptotic DNA fragments, namely In Situ Hybridization Chain Reaction, isHCR, was established. The principle of the assay is that the sticky end sequence of the apoptotic cell DNA fragment non-specifically initiates a hybridization chain reaction that specifically detects the apoptotic cell. The results of the combined TUNEL and isHCR method demonstrated that the majority of isHCR-positive cells were also labeled by TUNEL. In situ HCR often detect DNA fragments in the cytoplasm that the classical TUNEL method couldnot, and these cells may be in the early stages of apoptosis. It also indicates that DNA fragments are transferred to the cytoplasm during apoptosis. Because the staining process does not require terminal deoxynucleotidyl transferase as TUNEL staining does, isHCR staining cost low and can be performed on a large number of tissue specimens. It is believed that isHCR has the potential to detect DNA fragmentation of apoptotic cells in situ.

A User's Guide to Golden Gate Cloning Methods and Standards.

The continual demand for specialized molecular cloning techniques that suit a broad range of applications has driven the development of many different cloning strategies. One method that has gained significant traction is Golden Gate assembly, which achieves hierarchical assembly of DNA parts by utilizing Type IIS restriction enzymes to produce user-specified sticky ends on cut DNA fragments. This technique has been modularized and standardized, and includes different subfamilies of methods, the most widely adopted of which are the MoClo and Golden Braid standards. Moreover, specialized toolboxes tailored to specific applications or organisms are also available. Still, the quantity and range of assembly methods can constitute a barrier to adoption for new users, and even experienced scientists might find it difficult to discern which tools are best suited toward their goals. In this review, we provide a beginner-friendly guide to Golden Gate assembly, compare the different available standards, and detail the specific features and quirks of commonly used toolboxes. We also provide an update on the state-of-the-art in Golden Gate technology, discussing recent advances and challenges to inform existing users and promote standard practices.

Label-free palindromic DNA nanospheres as naked-eye colorimetric assay platform for detection of telomerase activity

A versatile naked-eye colorimetric assay platform is a desirable format for rapid analysis of disease-specific biomarkers, but is constrained by a complicated labeling process and false-positive signal readout. Target-triggered label-free palindromic DNA nanospheres (P-DNANS) were developed for the first time for the visual detection of telomerase activity. Telomerase recognition and extension trigger a hybridization event that yields Y-shaped double helices as core motifs. Y-shaped DNAs were linked by their palindromic sticky ends and P-DNANS were subsequently assembled with SYBR Green I (SG) in the helix. The SG-induced aggregation of gold nanoparticles is inhibited, and thus the red color can be observed by the naked eye. This label-free platform offers a cost-effective method for the cancer diagnosis and identification of breast cancer phenotypes.