Sticky Ends Research Articles

Artificial Water Channels—Progress Innovations and Prospects

Artificial Water Channels—Progress Innovations and Prospects

Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies.

Atomic Force Microscopy (AFM) is widely used for topographic imaging of DNA and protein-DNA complexes in ambient conditions with nanometer resolution. In AFM studies of protein-DNA complexes, identifying the protein’s location on the DNA substrate is one of the major goals. Such studies require distinguishing between the DNA ends, which can be accomplished by end-specific labeling of the DNA substrate. We selected as labels three-way DNA junctions (3WJ) assembled from synthetic DNA oligonucleotides with two arms of 39–40 bp each. The third arm has a three-nucleotide overhang, GCT, which is paired with the sticky end of the DNA substrate generated by the SapI enzyme. Ligation of the 3WJ results in the formation of a Y-type structure at the end of the linear DNA mole cule, which is routinely identified in the AFM images. The yield of labeling is 69%. The relative orientation of arms in the Y-end varies, such dynamics were directly visualized with time-lapse AFM studies using high-speed AFM (HS-AFM). This labeling approach was applied to the characterization of the nucleosome arrays assembled on different DNA templates. HS-AFM experiments revealed a high dynamic of nucleosomes resulting in a spontaneous unraveling followed by disassembly of nucleosomes.

CRISPR-Cas12a-activated palindrome-catalytic hairpin assembly for ultrasensitive fluorescence detection of HIV-1 DNA

Accurate analysis of HIV DNA is valuable for the diagnosis of AIDS. Herein, an ultrasensitive and specific fluorescence method was developed for HIV-1 DNA detection based on CRISPR-Cas12a-activated palindrome-catalytic hairpin assembly (CRISPR-Cas12a-PCHA). The presence of HIV-1 DNA activated the trans-cleavage activity of CRISPR-Cas12a, which could continuously digest the DNA fragment of hairpins connected to magnetic beads to expose single-stranded RNA. After magnetic separation, the exposed RNA triggered multiple PCHA reactions, generating many Y-shaped DNA structures that were self-assembled into the DNA superstructures via the hybridization of palindromic sticky ends, leading to the release of amounts of fluorescence signal. Different from the reported recently biosensing strategies of nucleic acid amplification technologies-activated CRISPR-Cas12a, CRISPR-Cas12a-PCHA endowed the strategy with unique advantages of simple sample pretreatment, direct duplex target detection, and ultrahigh sensitivity. The strategy was able to resist the interference of the complex matrix in real sample and distinguish between HIV patients and healthy persons. Thus, the method is a promising tool for ultrasensitive and specific detection of HIV-1 DNA for AIDS diagnosis.

Nanoscale packing of DNA tiles into DNA macromolecular lattices

Nanoscale double-crossovers (DX), antiparallel (A), and even half-turns-perimeter (E) DNA tiles (DAE-tiles) with rectangular shapes can be packed into large arrays of micrometer-scale lattices. But the features and mechanical strength of DNA assembly made from differently shaped large-sized DAE DNA tiles and the effects of various geometries on the final DNA assembly are yet to be explored. Herein, we focused on examining DNA lattices synthesized from DX bi-triangular, DNA tiles (T) with concave and convex regions along the perimeter of the tiles. The bi-triangular DNA tiles “T(A) and T(B)” were synthesized by self-assembling the freshly prepared short circular scaffold (S) strands “S(A) and S(B)”, each of 106 nucleotides (NT) lengths. The tiles “T(A) and T(B)” were then coupled together to get assembled via sticky ends. It resulted in the polymerization of DNA tiles into large-sized DNA lattices with giant micrometer-scale dimensions to form the “T(A) + T(B)” assembly. These DNA macro-frameworks were visualized “in the air” under atomic force microscopy (AFM) employing tapping mode. We have characterized how curvature in DNA tiles may undergo transitions and transformations to adjust the overall torque, strain, twists, and the topology of the final self-assembly array of DNA tiles. According to our results, our large-span DX tiles assembly “T(A) + T(B)” despite the complicated curvatures and mechanics, was successfully packed into giant DNA lattices of the width of 30–500 nm and lengths of 500 nm to over 10 μm. Conclusively, the micrometer-scale “T(A) + T(B)” framework assembly was rigid, stable, stiff, and exhibited enough tensile strength to form monocrystalline lattices.

Graph Computation Using Algorithmic Self-Assembly of DNA Molecules.

DNA molecules have been used as novel computing tools, by which Synthetic DNA was designed to execute computing processes with a programmable sequence. Here, we proposed a parallel computing method using DNA origamis as agents to solve the three-color problem, an example of the graph problem. Each agent was fabricated with a DNA origami of ∼50 nm diameter and contained DNA probes with programmable sticky ends that execute preset computing processes. With the interaction of different nanoagents, DNA molecules self-assemble into spatial nanostructures, which embody the computation results of the three-color problem with polynomial numbers of computing nanoagents in a one-pot annealing step. The computing results were confirmed by atomic force microscopy. Our method is completely different from existing DNA computing methods in its computing algorithm, and it has an advantage in terms of computational complexity and results detection for solving graph problems.

Powering ≈50 µm Motion by a Molecular Event in DNA Crystals.

A major challenge in material design is to couple nanoscale molecular and supramolecular events into desired chemical, physical, and mechanical properties at the macroscopic scale. Here, a novel self-assembled DNA crystal actuator is reported, which has reversible, directional expansion and contraction for over 50 μm in response to versatile stimuli, including temperature, ionic strength, pH, and redox potential. The macroscopic actuation is powered by cooperative dissociation or cohesion of thousands of DNA sticky ends at the designed crystal contacts. The increase in crystal porosity and cavity in the expanded state dramatically enhances the crystal capability to accommodate/encapsulate nanoparticles/proteins, while the contraction enables a "sponge squeezing" motion for releasing nanoparticles. This crystal actuator is envisioned to be useful for a wide range of applications, including powering self-propelled robotics, sensing subtle environmental changes, constructing functional hybrid materials, and working in drug controlled-release systems.

Optimization and performance evaluation of double-stranded probe in real-time PCR

BackgroundTaqMan probe-based real-time PCR (qPCR/RT-qPCR) has been widely used in various fields because of its high sensitivity and specificity. However, TaqMan probes are associated with a relatively higher background signal, and hence negatively affect the detection results. MethodsDouble-stranded probes (DSPs) were designed for the high sensitive detection of hepatitis B virus (HBV) DNA and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA using qPCR/RT-qPCR. Old DSPs (ODSPs) consist of different lengths of positive and negative strands with complementary oligonucleotides. We systematically optimized ODSPs length, the free energy of hybridization (ΔG) between complementary oligonucleotides, and the length of sticky ends, and the novel DSPs performances were evaluated in comparison with other types of probes. ResultsBy using similar length positive and negative strands, controlling ΔG between complementary oligonucleotides to approximately −30 kcal/mol, and maintaining the sticky end length at 4–6 nt, the analytical performances of DSPs were significantly improved. Compared with other types of probes, DSPs are advantageous in fluorescence signal intensity and sensitivity. ConclusionDSPs can further improve the detection sensitivity and the detection rate of low-concentration samples in molecular diagnosis.

Programming DNA Self-Assembly by Geometry†.

This manuscript introduces geometry as a means to program the tile-based DNA self-assembly in two and three dimensions. This strategy complements the sequence-focused programmable assembly. DNA crystal assembly critically relies on intermotif, sticky-end cohesion, which requires complementarity not only in sequence but also in geometry. For DNA motifs to assemble into crystals, they must be associated with each other in the proper geometry and orientation to ensure that geometric hindrance does not prevent sticky ends from associating. For DNA motifs with exactly the same pair of sticky-end sequences, by adjusting the length (thus, helical twisting phase) of the motif branches, it is possible to program the assembly of these distinct motifs to either mix with one another, to self-sort and consequently separate from one another, or to be alternatingly arranged. We demonstrate the ability to program homogeneous crystals, DNA "alloy" crystals, and definable grain boundaries through self-assembly. We believe that the integration of this strategy and conventional sequence-focused assembly strategy could further expand the programming versatility of DNA self-assembly.

Catalytic hairpin activated gold-magnetic/gold-core-silver-shell rapid self-assembly for ultrasensitive Staphylococcus aureus sensing via PDMS-based SERS platform

Staphylococcus aureus (S. aureus) has been identified as a marker of food contamination, closely associated with human health. This work designs a sensitive and rapid bio-detection strategy for S. aureus based on hybridization chain reaction-assisted surface enhanced Raman scattering (HCR-assisted-SERS) signal amplification. In this approach, the interaction between the aptamer (Apt) and its partial complementary DNA strands (cDNA) fabricated on the surface of gold-assisted magnetic nanoparticles (Au-MNPs) and the subsequent detachment of the cDNA results in the activation of the HCR process. In the HCR, a pair of hairpin structured DNA probes (H1 and H2) with sticky ends self-assembles to form a long DNA polymer. Subsequently, the output and amplification of the SERS signal were performed by conjugating 4-ATP modified Au@Ag NPs with the obtained DNA polymer via a specific Ag–S bond, and further collected through a self-administered polydimethylsiloxane (PDMS) cone-shaped support array. The precise quantification of S. aureus was performed in the concentration range of 28 to 2.8 × 106 cfu/mL, achieving a detection limit of 0.25 cfu/mL. This strategy was further applied to S. aureus detection in spiked milk samples with good recoveries (91–102%) and the relative standard deviation (4.35–8.41%). The sensing platform also showed satisfactory validation results (p > 0.05) using the traditional plate counting method. The proposed HCR-assisted SERS probe can be extended to other foodborne pathogenic bacteria types via engineering appropriate Apt and DNA initiators, thus, inspiring widespread applications in food safety and biomedical research.

Reinforcing DNA Supramolecular Hydrogel with Polymeric Multiple-Unit Linker

Reinforcing DNA Supramolecular Hydrogel with Polymeric Multiple-Unit Linker

Colorimetric aptasensor for on-site detection of acetamiprid with hybridization chain reaction-assisted amplification and smartphone readout strategy

An instruments-free colorimetric assay is established for acetamiprid detection with hybridization chain reaction (HCR)-based amplification strategy. Hairpin probes help gold nanoparticles (AuNPs) to resist salt-induced aggregation through sticky ends. In the presence of acetamiprid, aptamer hairpin probe HP opens due to the specifical recognition, then triggers an explosive hybridization between hairpin probes HA-1/2 and numerous hairpin probes are consumed. With the depletion of hairpin probes, the protection weakens and AuNPs suffer from salt-induced aggregation, which results in a color change from pinkish purplish to blue-purple. Under optimal conditions, the assay exhibits high sensitivity for acetamiprid analysis with a detection limit at 1.74 μM. Meanwhile, the aptasensor displays excellent selectivity that only acetamiprid can trigger the HCR and achieve enhanced detection. Worthwhile, based on the color variation, a smartphone readout-based strategy is proposed for visual quantification of acetamiprid, and good practical performance is obtained in real agricultural samples. The above visual method is portable and economic that get rid of advanced instruments, and shows a promising application prospect for on-site detection of acetamiprid.

Ligation-assisted homologous recombination enables precise genome editing by deploying both MMEJ and HDR.

CRISPR/Cas12a is a single effector nuclease that, like CRISPR/Cas9, has been harnessed for genome editing based on its ability to generate targeted DNA double strand breaks (DSBs). Unlike the blunt-ended DSB generated by Cas9, Cas12a generates sticky-ended DSB that could potentially aid precise genome editing, but this unique feature has thus far been underutilized. In the current study, we found that a short double-stranded DNA (dsDNA) repair template containing a sticky end that matched one of the Cas12a-generated DSB ends and a homologous arm sharing homology with the genomic region adjacent to the other end of the DSB enabled precise repair of the DSB and introduced a desired nucleotide substitution. We termed this strategy ‘Ligation-Assisted Homologous Recombination’ (LAHR). Compared to the single-stranded oligo deoxyribonucleotide (ssODN)-mediated homology directed repair (HDR), LAHR yields relatively high editing efficiency as demonstrated for both a reporter gene and endogenous genes. We found that both HDR and microhomology-mediated end joining (MMEJ) mechanisms are involved in the LAHR process. Our LAHR genome editing strategy, extends the repertoire of genome editing technologies and provides a broader understanding of the type and role of DNA repair mechanisms involved in genome editing.

Building Polyvalent DNA-Functionalized Anisotropic AuNPs using Poly-Guanine-Mediated In-Situ Synthesis for LSPR-Based Assays: Case Study on OncomiR-155.

DNA-functionalized gold nanoparticles (DNA-AuNPs) hold great promise for numerous biomedical applications, especially the building of well-defined nanosystems. Previously reported methods for the preparation of DNA-AuNPs all rely on the use of DNA-bearing free thiol or disulfide groups at their 3'/5' ends. But here we report a novel polyvalent DNA-AuNPs conjugation approach by in-situ fast synthesis of AuNPs at the polyguanine (G12 ) strands. As confirmed by both TEM images and gel electrophoresis analysis, many poly G strand can form an individual anisotropic AuNP and so each AuNP functionalized with a dense layer of DNA, resulting in the formation of polyvalent (p)DNA-AuNPs. The general applicability of this novel approach was further verified in hybridization test and UV-Vis spectroscopy results show that pDNA-AuNPs conjugation is more attractive in biomedical diagnosis and specific sequence detection like microRNA-155 by using an extra-strand poly G with "sticky end" that are complementary to the target sequence.

Another View of Weakly Open Sets Via DNA Recombination

The generalized structure of deoxyribonucleic acid (DNA) is based on the rules of topological spaces. DNA recombination is one of the most important processes within DNA, as it is essential in the pharmaceutical industry as well as in gene therapy. In this paper, we are discussing the relationship between rough sets, nano topological spaces (N), nano Z open (N) sets, and DNA recombination. We also created a new recombination mapping using the properties of the DNA recombination process. Further, by using the process of cutting and sticking of a sequence of genes, new topological structures are constructed and some of their properties and characterizations are investigated. Moreover, we study recombination operators in the statement “Sticky Ends”. Furthermore, we use nano topological structures to prove the validity of the mathematical model of the recombination process and the extent to which the topological mathematical properties correspond to the biological properties. Finally, we use nano Z-open sets to study many topological characteristics of the neighborhood, closure, interior, limit points, frontier, border and exterior.

Tetraphenylethylene-DNA conjugates: influence of sticky ends and DNA sequence length on the supramolecular assembly of AIE-active vesicles.

The supramolecular assembly of DNA conjugates, functionalized with tetraphenylethylene (TPE) sticky ends, into vesicular structures is described. The aggregation-induced emission (AIE) active TPE units allow monitoring the assembly process by fluorescence spectroscopy. The number of TPE modifications in the overhangs of the conjugates influences the supramolecular assembly behavior. A minimum of two TPE residues on each end are required to ensure a well-defined assembly process. The design of the presented DNA-based nanostructures offers tailored functionalization with applications in DNA nanotechnology.

Biomedical DNA hydrogels

Due to considerable progress in DNA nanotechnology, DNA is gaining significant attention as a programmable building block for the next generation of soft biomaterials. DNA has been used as either the only component to form all-DNA hydrogels or a cross-linker or functional entity to form hybrid DNA hydrogels through physical interactions or chemical reactions. The formed hydrogels exhibit adequate biocompatibility, convenient programmability, tunable multifunctionality, and capability of precise molecular recognition, making them an irreplaceable polymeric platform for interfacing biology. Responsive DNA hydrogels that are prepared through hybridization of DNA sticky ends, formation of i-motifs, enzymatic ligation, and enzymatic polymerization are commonly reported nowadays, which can undergo disassembly induced by various triggers, including alteration in ionic strength, pH, temperature, and biomolecules. These hydrogels are envisioned for applications of drug delivery and biosensing. This perspective aims to assess the most recent and important developments in this emerging class of biomedically useful DNA hydrogels.

単分散GUV内での均一DNAゲル形成

We attempted to control the generation and resultant size of DNA condensates (DNA gel and DNA droplet) comprised of sequence-designed ssDNA encapsulated in monodisperse giant unilamellar vesicles (GUVs) fabricated using microfluidic channels. By applying the osmotic shrinkage of GUVs, the internal contents including DNA nanostructures and salts were gradually concentrated to form DNA condensates. We investigated the effects of important parameters such as the length of sticky ends, DNA concentration, and salinity on the formation of DNA condensates. We found conditions under which the DNA condensates developed and grew as the GUV shrunk at room temperature. The present approach enabled the formation of uniform DNA condensates within a cell-sized lipid vesicle as a mimic of a nucleus, providing a method to construct the hierarchical structure of artificial cells.

Ladder-Like DNA Nanostructure-Mediated Cascade Catalytic Nanomachine for Construction of Ultrasensitive Biosensors.

Herein, a novel two-dimensional ladder-like DNA nanostructure (LDN)-mediated cascade catalytic nanomachine (LDN-CCN) with a higher catalytic efficiency beyond those of a conventional one-dimensional hybrid chain reaction (HCR) nanostructure-mediated CCN was constructed and applied to design an electrochemical biosensing platform with first-rank performance for ultrasensitive detection of target miRNA-21. First, output DNA S1' and S2' were produced through the DNAzyme recycle amplification when the target miRNA-21 existed. Then, the controllable LDN-CCN was constructed on S1-S2 modified electrodes by the subsequent reaction triggered by S1' and S2' with H1-AuNPs, H2, H3-AuNPs, and H4 with the assistance of K+ and hemin, in which the hemin/G-quadruplexes could produce a prominent electrochemical signal response to the substrate glucose. The best performance of cascade catalysis was acquired when the distance of Au nanoparticles (glucose oxidase-like activity) modified on H1 and H3 and hemin/G-quadruplexes (peroxidase-like activity) formed by the sticky ends of H2 and H4 was roughly 9 nm (27 bp) in LDN-CCN. The constructed electrochemical platform realized the sensitive detection of target miRNA-21 with the linear range from 100 aM to 10 nM and with a detection limit as low as 48.5 aM, which provided novel insights to explore the new functional DNA nanostructure and well-performing mimic enzyme cascade catalytic platforms for applications in biological fields and early diagnosis of diseases.

Development of a CRISPR/Cpf1 gene editing system in silkworm Bombyx mori

The CRISPR/Cas9 gene editing system has been widely used in basic research, gene therapy and genetic engineering due to its high efficiency, fast speed and convenience. Meanwhile, the discovery of novel CRISPR/Cas systems in the microbial community also accelerated the emergence of novel gene editing tools. CRISPR/Cpf1 is the second type (V type) CRISPR system that can edit mammalian genome. Compared with the CRISPR/Cas9, CRISPR/Cpf1 can use 5'T-PAM rich region to increase the genome coverage, and has many advantages, such as sticky end of cleavage site and less homologous recombination repair. Here we constructed three CRISPR/Cpf1 (AsCpf1, FnCpf1 and LbCpf1) expression vectors in silkworm cells. We selected a highly conserved BmHSP60 gene and an ATPase family BmATAD3A gene to design the target gRNA, and constructed gHSP60-266 and gATAD3A-346 knockout vectors. The efficiency for editing the target genes BmATAD3A and BmHSP60 by AsCpf1, FnCpf1 and LbCpf1 were analyzed by T7E1 analysis and T-clone sequencing. Moreover, the effects of target gene knockout by different gene editing systems on the protein translation of BmHSP60 and BmATAD3A were analyzed by Western blotting. We demonstrate the CRISPR/Cpf1 gene editing system developed in this study could effectively edit the silkworm genome, thus providing a novel method for silkworm gene function research, genetic engineering and genetic breeding.

Target DNA- and pH-responsive DNA hydrogel-based capillary assay for the optical detection of short SARS-CoV-2 cDNA.

DNA is recognized as a powerful biomarker for clinical diagnostics because its specific sequences are closely related to the cause and development of diseases. However, achieving rapid, low-cost, and sensitive detection of short-length target DNA still remains a considerable challenge. Herein, we successfully combine the catalytic hairpin assembly (CHA) technique with capillary action to develop a new and cost-effective method, a target DNA- and pH-responsive DNA hydrogel–based capillary assay, for the naked eye detection of 24 nt short single-stranded target DNA. Upon contact of target DNA, three individual hairpin DNAs hybridize with each other to sufficiently amplify Y-shaped DNA nanostructures (Y-DNA) until they are completely consumed via CHA cycling reactions. Each arm of the resultant Y-DNA contains sticky ends with i-motif DNA structure-forming sequences that can be self-assembled in an acidic environment (pH 5.0) to form target DNA- and pH-responsive DNA hydrogels by means of i-motif DNA-driven crosslinking. When inserting a capillary tube in the resultant solution, the liquid level inside clearly reduces due to the decrease in capillary force induced by the gels. In this way, the developed assay demonstrates sensitive and quantitative detection, with a detection limit of approximately 10 pM of 24 nt short complementary DNA (cDNA) targeting SARS-CoV-2 RNA genes at room temperature within 1 h. The assay is further shown to successfully detect target cDNA in serum, and it is also applied to detect several types of target sequences. Requiring no analytic equipment, precise temperature control, or enzymatic reactions, the developed DNA hydrogel–based capillary assay has potential as a promising naked eye detection platform for target DNA in resource-limited clinical settings.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00604-021-05138-9.