DNA Three-way Junction Research Articles

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.

Heterogeneous migration routes of DNA triplet repeat slip-outs

It is unclear how the length of a repetitive DNA tract determines the onset and progression of repeat expansion diseases, but the dynamics of secondary DNA structures formed by repeat sequences are believed to play an important role. It was recently shown that three-way DNA junctions containing slip-out hairpins of CAG or CTG repeats and contiguous triplet repeats in the adjacent duplex displayed single-molecule FRET (smFRET) dynamics that were ascribed to both local conformational motions and longer-range branch migration. Here we explore these so-called "mobile" slip-out structures through a detailed kinetic analysis of smFRET trajectories and coarse-grained modeling. Despite the apparent structural simplicity, with six FRET states resolvable, most smFRET states displayed biexponential dwell-time distributions, attributed to structural heterogeneity and overlapping FRET states. Coarse-grained modeling for a (GAC)10 repeat slip-out included trajectories that corresponded to a complete round of branch migration; the structured free energy landscape between slippage events supports the dynamical complexity observed by smFRET. A hairpin slip-out with 40 CAG repeats, which is above the repeat length required for disease in several triplet repeat disorders, displayed smFRET dwell times that were on average double those of 3WJs with 10 repeats. The rate of secondary-structure rearrangement via branch migration, relative to particular DNA processing pathways, may be an important factor in the expansion of triplet repeat expansion diseases.

Bipyridine-Modified DNA Three-Way Junctions with Amide Linkers: Metal-Dependent Structure Induction and Self-Sorting.

Invited for the cover of this issue are Yusuke Takezawa, Shiori Sakakibara, and Mitsuhiko Shionoya at The University of Tokyo. The image depicts the formation of stable DNA three-way junction structures crosslinked by an interstrand NiII (bpy)3 complex. Read the full text of the article at 10.1002/chem.202104037.

A novel aptasensor for colorimetric monitoring of tobramycin: Strategy of enzyme-like activity of AuNPs controlled by three-way junction DNA pockets

In this study, a novel colorimetric sensor was introduced to detect tobramycin (TOB) based on controlling the catalytic activity of gold nanoparticles (AuNPs) by the three-way junction aptamer pockets. In the absence of TOB, the surfaces of AuNPs were masked by the three-way junction pockets that prevented their catalytic activation for the reduction of 4-Nitrophenol in the presence of NaBH4. While the formation of the pockets was prevented in the presence of TOB that facilitated the 4-Nitrophenol access to AuNPs. Hence, the catalytic reduction of 4-Nitrophenol induced a color change of the solution from yellow to colorless, highlighting the presence of the target. The aptasensing assay provided good target specificity with a detection limit (LOD) of 1.16 µM and a linear dynamic range over 4–32 µM. The aptasensor was successfully applied to quantitatively monitor TOB in the human serum and milk samples with the LODs of 1.38 and 1.42 µM and recovery values of 94.87–105.75% and 93.75–105.31%, respectively.

Front Cover: Bipyridine‐Modified DNA Three‐Way Junctions with Amide linkers: Metal‐Dependent Structure Induction and Self‐Sorting (Chem. Eur. J. 67/2021)

A bipyridine-modified DNA three-way junction (3WJ) structure has been synthesized from three DNA strands modified with a bipyridine (bpy) ligand via a stable amide linker. The bpy-modified 3WJ was thermally stabilized by the formation of an interstrand NiII(bpy)3 complex. Structural induction of the 3WJs and the duplexes was achieved by adding or removing NiII ions. NiII-mediated self-sorting of 3WJs was also demonstrated by using the bpy-modified strands and their unmodified counterparts. More information can be found in the Full Paper by Y. Takezawa, S. Sakakibara, and M. Shionoya (DOI: 10.1002/chem.202102977).

Bipyridine-Modified DNA Three-Way Junctions with Amide linkers: Metal-Dependent Structure Induction and Self-Sorting.

DNA three-way junction (3WJ) structures are essential building blocks for the construction of DNA nanoarchitectures. We have synthesized a bipyridine (bpy)-modified DNA 3WJ by using a newly designed bpy-modified nucleoside, Ubpy -3, in which a bpy ligand is tethered via a stable amide linker. The thermal stability of the bpy-modified 3WJ was greatly enhanced by the formation of an interstrand NiII (bpy)3 complex at the junction core (ΔTm =+17.7 °C). Although the stereochemistry of the modification site differs from that of the previously reported bpy-modified nucleoside Ubpy -2, the degree of the NiII -mediated stabilization observed with Ubpy -3 was comparable to that of Ubpy -2. Structure induction of the 3WJs and the duplexes was carried out by the addition or removal of NiII ions. Furthermore, NiII -mediated self-sorting of 3WJs was performed by using the bpy-modified strands and their unmodified counterparts. Both transformations were driven by the formation of NiII (bpy)3 complexes. The structural induction and self-sorting of bpy-modified 3WJs are expected to have many potential applications in the development of metal-responsive DNA materials.

Dual targeting of higher-order DNA structures by azacryptands induces DNA junction-mediated DNA damage in cancer cells.

DNA is intrinsically dynamic and folds transiently into alternative higher-order structures such as G-quadruplexes (G4s) and three-way DNA junctions (TWJs). G4s and TWJs can be stabilised by small molecules (ligands) that have high chemotherapeutic potential, either as standalone DNA damaging agents or combined in synthetic lethality strategies. While previous approaches have claimed to use ligands that specifically target either G4s or TWJs, we report here on a new approach in which ligands targeting both TWJs and G4s in vitro demonstrate cellular effects distinct from that of G4 ligands, and attributable to TWJ targeting. The DNA binding modes of these new, dual TWJ-/G4-ligands were studied by a panel of in vitro methods and theoretical simulations, and their cellular properties by extensive cell-based assays. We show here that cytotoxic activity of TWJ-/G4-ligands is mitigated by the DNA damage response (DDR) and DNA topoisomerase 2 (TOP2), making them different from typical G4-ligands, and implying a pivotal role of TWJs in cells. We designed and used a clickable ligand, TrisNP-α, to provide unique insights into the TWJ landscape in cells and its modulation upon co-treatments. This wealth of data was exploited to design an efficient synthetic lethality strategy combining dual ligands with clinically relevant DDR inhibitors.

DNA Three-Way Junction with Multiple Recognition Regions Mediated an Unconfined DNA Walker for Electrochemical Ultrasensitive Detection of miRNA-182-5p.

In this work, a DNA three-way junction (TWJ) with multiple recognition regions was intelligently engineered, which could be applied as an unconfined DNA walker with a rapid walking speed and high sensitivity for electrochemical detection of microRNA (miRNA-182-5p). Once the target miRNA was presented, the hairpins on TWJ could be successively opened to form an annular DNA walker, which could walk on the entire scope of the electrode surface without the confine for the length of DNA walker legs compared with the traditional DNA walker, greatly improving the walking efficiency. In addition, this DNA walker with multirecognition segments could obviously increase the local concentration of recognition sites, which significantly enhanced the detection speed and sensitivity. As a result, this proposed biosensor with annular DNA as a walker could dexterously achieve the ultrasensitive and fast detection of miRNA-182-5p from 0.1 fM to 1 nM with a detection limit of 31.13 aM. Meaningfully, this strategy explored an innovative path in the design of a new DNA walker nanostructure for accomplishing speedy and sensitive detection of biomarkers.

Visible Fluorescent Light-up Probe for DNA Three-Way Junctions Provides Host-Guest Biosensing Applications.

DNA three-way junctions (3WJs) consist of a Y-shaped hydrophobic branch point connecting three double-stranded stems and are viewed as druggable targets for cancer treatment. They are also important building blocks for the construction of DNA nanostructures and serve as recognition elements for DNA aptasensors for a wide variety of diagnostic applications. However, visible fluorescent light-up probes for specific staining of DNA 3WJs are currently lacking. Herein, we report that a merocyanine containing the N-methylbenzothiazolium (Btz) acceptor vinyl linked to a 2-fluorophenolic (FPhO) donor (FPhOBtz) serves as a universal fluorescent turn-on dye for DNA 3WJs. Our evidence is based on a multifaceted approach to define the specificity and affinity of FPhOBtz for 3WJ DNA aptamers; the cocaine binding aptamer MN4, the cholic acid binding aptamer (CABA), and four steroid aptamers (DOGS.1, DISS.1, BES.1, DCAS.1). FPhOBtz exhibits impressive turn-on (up to 730-fold) fluorescence at 580 nm upon aptamer binding with low micromolar affinity. Direct FPhOBtz displacement from the 3WJ binding domain through competitive alkaloid and steroid binding provides immediate fluorescent read out for host-guest detection strategies in human blood serum in the low micromolar regime. Our results present the first visible light-up fluorescent probe for DNA 3WJ detection strategies.

Fabrication of a surface-enhanced Raman spectroscopy-based analytical method consisting of multifunctional DNA three-way junction-conjugated porous gold nanoparticles and Au-Te nanoworm for C-reactive protein detection.

C-Reactive protein (CRP) is a biomarker of inflammatory responses and an index for assessing the risk of cardiovascular disease and estimating prognosis. In this study, we constructed a surface-enhanced Raman spectroscopy (SERS) biosensor composed of a multifunctional DNA three-way junction (DNA 3WJ), porous gold nanoplates (pAuNPs), and an Au-Te nanoworm structure for detection of CRP. The pAuNP and Au-Te nanostructures were synthesized by galvanic replacement reactions, and the morphology was confirmed by transmission electron microscopy, scanning electron microscopy, and dynamic light scattering (DLS). To generate the SERS signal, the Au-Te nanostructure was immobilized on an indium-tin oxide substrate, and the thiol-modified CRP aptamer was then self-assembled onto the modified substrate for CRP recognition. To amplify the SERS signal and identify the Raman tag, the multifunctional DNA 3WJ was conjugated with the pAuNPs, and each fragment of 3WJ was functionalized to biotin (pAuNP conjugation), methylene blue (Raman reporter), and CRP aptamer (target binding). The results were confirmed by gel electrophoresis. For conjugation between pAuNPs and DNA 3WJ, avidin was encapsulated in pAuNPs, and the conjugation structure was confirmed by DLS. The fabricated SERS biosensor showed detection limits of 2.23 pM in phosphate-buffered saline and 3.11 pM in diluted human serum. Overall, the proposed biosensor may have potential applications as a SERS biosensor platform.

Stereoselective Self-Assembly of DNA Binding Helicates Directed by the Viral β-Annulus Trimeric Peptide Motif.

Combining coordination chemistry and peptide engineering offers extraordinary opportunities for developing novel molecular (supra)structures. Here, we demonstrate that the β-annulus motif is capable of directing the stereoselective assembly of designed peptides containing 2,2′-bipyridine ligands into parallel three-stranded chiral peptide helicates, and that these helicates selectively bind with high affinity to three-way DNA junctions.

Fabrication of an Electrochemical Aptasensor Composed of Multifunctional DNA Three-Way Junction on Au Microgap Electrode for Interferon Gamma Detection in Human Serum.

Interferon gamma (IFN-γ) is an important cytokine with antiviral, antibacterial, and immunosuppressive properties. It has been used as a biomarker for the early detection of several diseases, including cancer, human immunodeficiency virus (HIV), tuberculosis, and paratuberculosis. In this study, we developed an electrochemical biosensor composed of multifunctional DNA 3WJ to detect IFN-γ level with high sensitivity. Each multifunctional triple-stranded aptamer (MF-3WJ) was designed to have an IFN-γ aptamer sequence, anchoring region (thiol group), and 4C–C (cytosine–cytosine) mismatch sequence (signal generation), which could introduce silver ions. To generate the electrochemical signal, four Ag+ ions were intercalated (3wj b-3wj c) in the 4C–C mismatch sequence. MF-3WJ was assembled through the annealing step, and the assembly of MF-3WJ was confirmed by 8% tris–boric–EDTA native polyacrylamide gel electrophoresis. The Au microgap electrode was manufactured to load sample volumes of 5 µL. The reliability of electrochemical biosensor measurement was established by enabling the measurement of seven samples from one Au microgap electrode. MF-3WJ was immobilized on the Au microgap electrode. Then, cyclic voltammetry and electrochemical impedance spectroscopy were performed to confirm the electrochemical properties of MF-3WJ. To test the electrochemical biosensor’s ability to detect IFN-γ, the limit of detection (LOD) and selectivity tests were performed by square wave voltammetry. A linear region was observed in the concentration range of 1 pg/mL–10 ng/mL of IFN-γ. The LOD of the fabricated electrochemical biosensor was 0.67 pg/mL. In addition, for the clinical test, the LOD test was carried out for IFN-γ diluted in 10% human serum samples in the concentration range of 1 pg/mL–10 ng/mL, and the LOD was obtained at 0.42 pg/mL.

Directional Propulsion of DNA Microspheres Based on Light-Induced Asymmetric Growth of Peptide Nanofibers.

Inspired by natural motors, synthetic motors powered by light have emerged as promising platforms for constructing artificial micro/nanorobots. As a concept of light-driven motors, we have previously reported propulsion of giant liposomes driven by light-induced peptide nanofiber growth on the surface, inspired by natural pathogens using external actin polymerization for their propulsion. However, their movement was nondirectional. Here, we used DNA microspheres (also known as nucleospheres) comprising DNA three-way junctions with self-complementary sticky ends as vehicles for directional propulsion by light-induced peptide nanofiber growth. By introducing a peptide-DNA conjugate connected by a photocleavage unit to the surface of nucleospheres, ultraviolet (UV) light irradiation induced the asymmetric peptide nanofiber growth on the surface. Nucleospheres exhibited directional movement away from the light source, showing negative phototaxis. This directional movement was maintained even after the light irradiation was ceased. Our phototactic system helps to better understand the mechanism of natural motors and construct bioinspired motors with controlled movement.

A low-background fluorescent aptasensor for acetamiprid detection based on DNA three-way junction-formed G-quadruplexes and graphene oxide.

A simple fluorescence detection platform has been established for acetamiprid assay based on DNA three-way junctions (TWJs), which can triple the fluorescence signal without any other amplification. It is designed with three single-stranded DNAs (ssDNA), each of which contains one-third or two-thirds of the G-quadruplex sequence at each end. Upon the addition of acetamiprid, the conformation of the aptamer-containing double-stranded DNA (dsDNA) changes from its original conformation and releases a strand of ssDNA. This ssDNA, with the other two ssDNAs, can assemble into DNA TWJs, and the three pairs of the branched ends of the DNA TWJs are adjacent to each other, allowing them to form three units of G-quadruplexes. Hence, the fluorescence of N-methyl mesoporphyrin IX (NMM) is lighted by the nascent G-quadruplexes. Graphene oxide (GO) is then added to minimize the detection background by absorbing the free NMM and non-target-induced ssDNA. The proposed strategy can assay acetamiprid in a wide linear range of 0-500nM with a detection limit of 5.73nM. More importantly, this assay platform demonstrates high potential for acetamiprid assay in food control and environmental monitoring.

Dynamic Stereoselection of Peptide Helicates and Their Selective Labeling of DNA Replication Foci in Cells*.

Although largely overlooked in peptide engineering, coordination chemistry offers a new set of interactions that opens unexplored design opportunities for developing complex molecular structures. In this context, we report new artificial peptide ligands that fold into chiral helicates in the presence of labile metal ions such as FeII and CoII . Heterochiral β-turn-promoting sequences encode the stereoselective folding of the peptide ligands and define the physicochemical properties of their corresponding metal complexes. Circular dichroism and NMR spectroscopy in combination with computational methods allowed us to identify and determine the structure of two isochiral ΛΛ-helicates, folded as topological isomers. Finally, in addition to the in-vitro characterization of their selective binding to DNA three-way junctions, cell-microscopy experiments demonstrated that a rhodamine-labeled FeII helicate was internalized and selectively stains DNA replication factories in functional cells.

Engineering couplings for exciton transport using synthetic DNA scaffolds

Summary Control over excitons enables electronic energy to be harnessed and transported for light harvesting and molecular electronics. Such control requires nanoscale precision over the molecular components. Natural light-harvesting systems achieve this precision through sophisticated protein machinery, which is challenging to replicate synthetically. Here, we introduce a DNA-based platform that spatially organizes cyanine chromophores to construct tunable excitonic systems. We synthesized DNA-chromophore nanostructures and characterized them with ensemble ultrafast and single-molecule spectroscopy and structure-based modeling. This synthetic approach facilitated independent control over the coupling among the chromophores and between the chromophores and the environment. We demonstrated that the coupling between the chromophores and the environment could enhance exciton transport efficiency, highlighting the key role of the environment in driving exciton dynamics. Control over excitons, as reported here, offers a path toward the development of designer nanophotonic devices.

Conformational and migrational dynamics of slipped-strand DNA three-way junctions containing trinucleotide repeats

Expansions of CAG/CTG trinucleotide repeats in DNA are the cause of at least 17 degenerative human disorders, including Huntington’s Disease. Repeat instability is thought to occur via the formation of intrastrand hairpins during replication, repair, recombination, and transcription though relatively little is known about their structure and dynamics. We use single-molecule Förster resonance energy transfer to study DNA three-way junctions (3WJs) containing slip-outs composed of CAG or CTG repeats. 3WJs that only have repeats in the slip-out show two-state behavior, which we attribute to conformational flexibility at the 3WJ branchpoint. When the triplet repeats extend into the adjacent duplex, additional dynamics are observed, which we assign to interconversion of positional isomers. We propose a branchpoint migration model that involves conformational rearrangement, strand exchange, and bulge-loop movement. This migration has implications for how repeat slip-outs are processed by the cellular machinery, disease progression, and their development as drug targets.

A highly sensitive fluorescence biosensor for detection of Staphylococcus aureus based on HCR-mediated three-way DNA junction nicking enzyme assisted signal amplification.

Sensitive and efficient monitoring of food-borne bacteria is of great importance for food safety control. Herein, a novel biosensor for highly sensitive detection of Staphylococcus aureus (S. aureus) was constructed by combining hybridization chain reaction (HCR) and nicking enzyme. Different from the upstream-downstream based circuit, the proposed biosensor integrated HCR circuit and three-way DNA junction nicking enzyme assisted signal amplification (3WJ-NEASA) into a virtuous circle of promotion. In the HCR-mediated 3WJ-NEASA sensing strategy, target DNA of S. aureus initiated the self-assembly between HCR hairpins (H1 and H2), which exposed the gap to capture molecular beacon (MB) and construct the 3WJ structure. Meanwhile, MB increased the stability of HCR nanowires and enhanced the efficiency of the HCR circuit, and thus more 3WJ-NEASA circuits were generated in HCR nanowires. Benefiting from the synergistic amplification coupling HCR and 3WJ-NEASA, this isothermal biosensor can detect as low as 6.7 pM of target DNA in one step within only 30 min. Furthermore, the HCR-mediated 3WJ-NEASA assay has been applied in the detection of S. aureus with a limit of detection (LOD) as low as 1.2 × 101 cfu mL-1, and has exhibited reliable practicability in spiked milk. It is the first time that a DNA biosensor combining HCR and 3WJ-NEASA for dual signal amplification was developed and has been adopted to the sensitive analysis of food-borne bacteria. Additionally, this strategy can serve as a universal platform for monitoring other analytes, and therefore possesses broad application prospects in food safety and environmental monitoring.

Label-free tri-luminophores electrochemiluminescence sensor for microRNAs detection based on three-way DNA junction structure

Abstract A label-free microRNAs (miRNAs) electrochemiluminescence sensor was developed based on the synergistically enhanced ECL signal using novel ECL nanomaterial assembled by tri-luminophores perylenetetracarboxylic acid (PTCA) graphite-like carbon nitride nanosheet (g-C3N4 NS) and graphene carbon quantum dots (GQDs). The ECL intensity of our proposed nanocomposites increased significantly, by taking advantages of the synergistic promotion ECL mechanism of PTCA g-C3N4 and GQDs. Meanwhile, a three-way junction (TWJ) structure was introduced into the sensor design, whose conformation can be specifically induced in the presence of target miRNA. Thus, the formation of TWJ structure can hinder the contact between the co-reactant Na2S2O8 in the solution phase and the luminescent substrate, which produce a quenched ECL signal. Our proposed ECL biosensor provided a highly sensitive detection of miRNA-21 with a detection limit of 96 aM ranging from 100 aM to 1 nM. Furthermore, this ECL sensing system was able to successfully distinguish the target RNA from its homologous family and presented a promising response in human real samples. With excellent properties, the proposed ECL biosensor provided an efficient and ultrasensitive method for miRNAs detection.

Three-way junction DNA based electrochemical biosensor for microRNAs detection with distinguishable locked nucleic acid recognition and redox cycling signal amplification

Herein, we present one three-way junction (TWJ) DNA based electrochemical biosensor for sensitive microRNAs detection with locked nucleic acid (LNA) as capture probe and enzyme-free redox cycling signal amplification strategy. LNA owns higher binding affinity and thermal stability compared to normal DNA sequence. In the presence of target microRNA (miRNA-21), novel TWJ structure can be specially triggered with the assistant of methylene blue (MB)-tagged DNA signal probe, thus producing an enhanced electrochemical current. Furthermore, a chemical reductant, tris (2-carboxyethyl) phosphine (TCEP), is adopted to lead a relayed electron transport between solution TCEP and TWJ-linked MB tag and amplified whole signal intensity. Based on the selectivity from target-induced LNA/DNA TWJ structure and enzyme-free MB cycling amplification strategy, the designed electrochemical biosensor exhibits sensitive miRNA-21 detection, with a linear range from 100 aM to 100 pM and a low detection limit of 77 aM. In addition, it can successfully discriminate the differences between the miRNA family members, presenting a promising response to the direct determination of target miRNA-21 in real biological fluids. Our work provides an alternative LNA-based electrochemical biosensor way in the area of miRNA diagnostics and clinical analysis without complicated enzyme/nanomaterials-assisted signal amplification.