DNA Three-way Junction Research Articles

Detection of Mg2+-dependent, coaxial stacking rearrangements in a bulged three-way DNA junction by single-molecule FRET

Three-way helical junctions (3WJs) arise in genetic processing, and they have architectural and functional roles in structured nucleic acids. An internal bulge at the junction core allows the helical domains to become oriented into two possible, coaxially stacked conformers. Here, the helical stacking arrangements for a series of bulged, DNA 3WJs were examined using ensemble fluorescence resonance energy transfer (FRET) and single-molecule FRET (smFRET) approaches. The 3WJs varied according to the GC content and sequence of the junction core as well as the pyrimidine content of the internal bulge. Mg2+ titration experiments by ensemble FRET show that both stacking conformations have similar Mg2+ requirements for folding. Strikingly, smFRET experiments reveal that a specific junction sequence can populate both conformers and that this junction undergoes continual interconversion between the two stacked conformers. These findings will support the development of folding principles for the rational design of functional DNA nanostructures.

Directed Self-Assembly of Trimeric DNA-Bindingchiral Miniprotein Helicates.

We propose that peptides are highly versatile platforms for the precise design of supramolecular metal architectures, and particularly, for the controlled assembly of helicates. In this context, we show that the bacteriophage T4 Fibritin foldon (T4Ff) can been engineered on its N-terminus with metal-chelating 2,2′-bipyridine units that stereoselectively assemble in the presence of Fe(II) into parallel, three-stranded peptide helicates with preferred helical orientation. Modeling studies support the proposed self-assembly and the stability of the final helicate. Furthermore, we show that these designed mini-metalloproteins selectively recognize three-way DNA junctions over double-stranded DNA.

FRET-based assay using a three-way junction DNA substrate to identify inhibitors of human cytomegalovirus pUL89 endonuclease activity

Human cytomegalovirus (HCMV) packages its viral genome using a multi-protein complex often called the terminase complex. The terminase complex promotes the packaging of a single copy of the double-stranded viral DNA genome by cleaving concatemeric genomic DNA during particle assembly. The endonuclease activity necessary to cleave the viral genomic DNA derives from a terminase complex protein, pUL89. The genome cleavage step is necessary for infectious particle assembly and is therefore an interesting target for antiviral intervention. We and others have previously described agarose gel- and enzyme-linked immunosorbent assay-based procedures to detect pUL89 endonuclease activity; however, these approaches were labor intensive and low-throughput. To develop a screening platform for compound libraries, we constructed a fluorescently-labeled three-way junction DNA that continuously reported pUL89 endonuclease activity and was suitable for high-throughput screening. In a pilot screen of 1280 compounds, we identified trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid and (2′Z, 3′E)-6-bromoindirubin-3′-oxime as hits with low micromolar half maximal inhibitory concentration (IC50) values. The latter hit also inhibited HCMV at a late step in virus replication with a 1 μM half maximal effective concentration (EC50). We describe here the development and validation of a sensitive fluorescence resonance energy transfer-based assay for high throughput screening that identified inhibitors of pUL89 endonuclease activity and virus replication.

Electrogenerated chemiluminescence biosensor for detection of mercury (II) ion via target-triggered manipulation of DNA three-way junctions

A new electrogenerated chemiluminescence (ECL) biosensor is fabricated for the determination of mental ion incorporating DNA three-way junction structure (DNA-TWJ). As a model system, Hg2+ was chosen as an analyte. The ECL biosensor was fabricated by covalently coupling Hg2+ special DNA-TWJ tagged with ruthenium (II) complex (Ru) (named TW/Ru-J1) to the surface of glassy carbon electrode that had been covalently modified with 4-aminobenzoic acid via electrochemical oxidations. Upon binding of Hg2+ to the TW/Ru-J1, the confirmation of TW/Ru-J1 changed and induced Ru away from surface of electrode and thus led to a low ECL signal. The signal linearly decreases with the concentration of Hg2+ in the range from 0.1 pM to 10 pM with a detection limit of 0.04 pM This study could be easily extended to various analytical platforms for the detection of many kinds of analytes or their interactions such as DNA/RNA, DNAzyme/target, aptamer/target, and antibody/antigen.

Amperometric DNA biosensor for Mycobacterium tuberculosis detection using flower-like carbon nanotubes-polyaniline nanohybrid and enzyme-assisted signal amplification strategy

The authors described a universal amperometric DNA biosensor for rapid and ultrasensitive detection of the specific IS6110 DNA sequence of Mycobacterium tuberculosis (MTB). Surprisingly, when tufted carbon nanotubes (CNTs) doped with polyaniline (PAN), a unique flower-like structure with large surface area, abundant active groups and efficient redox activity was obtained. Accordingly, CNTs-PAN nanohybrid was designed both as novel redox nanoprobe and nanocarrier to decorate signal probe, forming tracer label for the generation and amplification of electrochemical signal. In addition, functionalized fullerene (C60) nanoparticles (FC60) were employed as sensor platform to accelerate electron transfer and increase the loading of capture probe (CP). Moreover, enzyme-assisted target recycling amplification including three-way DNA junction could resolve the restriction of target sequences and make this method universal for other analytes. In the presence of target DNA, the assistant probe (AP), together with target DNA, could hybridize with CP and open its hairpin structure to form the Y-shaped junction and recognition sequence for nicking endonuclease. Once the CP was cleaved, the released AP and target DNA could hybridize with another CP to trigger the next cycle of cleavage, resulting in a large number of cleaved CP. After hybridization between cleaved CP and tracer label, a significantly enhanced electrochemical signal of CNTs-PAN could be easily read out. Based on the multiple signal amplification strategy, a wide detection linear range from 1 fM to 10 nM was obtained for target DNA of MTB. More importantly, the universal DNA biosensor also showed high specificity and sensitivity for MTB detection in clinical samples, which may provide a pragmatic tool for MTB testing and hold a great potential for other analytes.

Three-way DNA junction structure combined with enzyme-powered cascade amplification for ultrasensitive electrochemiluminescence detection of microRNA via smart DNA walker

In this work, a target-triggered three-way junction (3-WJ) structure was combined with enzyme-powered cascade amplification strategy for ultrasensitive electrochemiluminescence (ECL) detection of microRNA via free-running DNA walker. In the presence of target microRNA-21 (miRNA-21), a three-way junction structure was formed to trigger enzyme-aided multiple DNA amplification, and thus exponentially amplified triggers were generated. Then, the trigger DNA could hybridize with the blocking DNA on the electrode to release walker DNA, which specifically combined with the quantum dots (QDs)-DNA probe, and formed a recognition site for Nt.BbvCI. The movement of DNA walker is powered by the nicking endonuclease that cleaves specific QDs-DNA probes on the track. Thus one walker DNA can automatically cleave multiple QDs probes on the electrode during the movement, resulting in significantly amplified changes of ECL signal, which could be used for the highly sensitive detection of target miRNA-21. Under the optimal conditions, the ECL signal change is linear with the concentration of miRNA-21 in the range from 10−14 M to 10-7 M with a detection limit of 1.5 fM. This newly established strategy could achieve rapid, isothermal, and homogeneous signal amplification for specific nucleic acids in complicated biomatrix, which hold great potential for application in early clinical diagnosis.

Three-way DNA junction based platform forultra-sensitive fluorometric detectionof multiple metal ions as exemplified for Cu(II), Mg(II) and Pb(II).

A DNA-based fluorometric method is described for simultaneous determination of multiple metal ions. It is based on recycling cleavage of hairpins by using a three-way DNA junction structure. Three DNA sequences containing a binding region and an enzyme-strand (E-DNA) region are hybridized to form a three-way DNA junction. The enzyme strand regions at the end of the DNA sequence binds to the substrate sequence (S-DNA) at the loop of the hairpin to form typical DNAzyme structures. In the presence of analyte metal ions, the DNAzyme structure thus formed cleaves the loop of hairpins. This is accompanied by a release of fluorescently labeled DNA fragments and by quenching of fluorescence. The detection limits are 35 pM for Cu(II), 2nM for Mg(II), and 8 pM for Pb(II). This method was successfully applied to the simultaneous determination of these ions in spiked human serum. Graphical abstract Schematic presentation of the recycling cleavage of hairpins by using a three-way DNA junction structure. It causes a release of fluorescently labeled DNA fragments and quenching of fluorescence. It was successfully applied to the simultaneous determination of Cu(II), Mg(II) and Pb(II) in spiked human serum.

DNA Directed Self-Assembly of Single Walled Carbon Nanotubes into Three-Way Junction Nanostructures.

Utilization of a self-assembled two-dimensional DNA nanostructure to arrange single-walled carbon nanotubes (SWNTs) into predetermined structures at controllable angles is presented. A specially designed DNA three-way junction (3WJ) composed of three double-stranded DNA arms containing single-stranded overhang sequences was prepared by annealing of partially complementary ssDNA sequences and ultrasonicated with SWNTs, resulting in DNA-3WJ/SWNT hybrid nanostructures. Utilization of DNA-3WJ not only allowed the precise dispersion of SWNTs but also acted as a rigid template for the self-assembly of SWNTs into three-armed junctions at an angle of approximately 120° to each other as visualized by scanning electron microscopy and atomic force microscopy. Prepared DNA-3WJ/SWNT nanostructures were also demonstrated to have the appropriate binding sites for fluorophores, providing a simple method for the fluorescent labeling of SWNTs. When ssDNA sequences forming the DNA-3WJ are ultrasonicated with SWNTs, followed by annealing of resulting ssDNA wrapped SWNTs, instead of hybrid junctions composed of three SWNT molecules, a web-like structure composed of interconnected SWNT junctions was obtained. The design approaches demonstrated here provide simple methods for the arrangement of SWNTs into desired nanostructures utilizing pre-assembled DNA nanostructures as linkers in aqueous solution through noncovalent interactions which can greatly contribute to efforts along the controlled assembly of SWNTs.

Supramolecular Recognition of Three Way Junction DNA by a Cationic Calix[3]carbazole

DNA three-way junctions (TWJ-DNA) are intermediate structures in DNA replication and/or recombination. They play very important roles in biological processes, but more subtle functions are still unknown due partially to the lack of a fluorescent ligand. In this study, a cationic calix[3]carbazole (2) has been synthesized and its properties of interacting with TWJ-DNA have been evaluated by UV/Vis and fluorescence spectroscopy, circular dichroism (CD), gel electrophoresis, and 1 H NMR studies. The results show that 2 binds to the central hydrophobic cavity of TWJ-DNA. Moreover, it could selectively bind to TWJ-DNA over duplex and quadruplex DNA. Furthermore, 2 possesses the capability of serving as the TWJ-DNA probe as its trap-II excimer emission is turned on by TWJ-DNA.

Sub-Ensemble Monitoring of DNA Strand Displacement Using Multiparameter Single-Molecule FRET.

Non-enzymatic DNA strand displacement is an important mechanism in dynamic DNA nanotechnology. Here, we show that the large parameter space that is accessible by single-molecule FRET is ideal for the simultaneous monitoring of multiple reactants and products of DNA strand exchange reactions. We monitored the strand displacement from double-stranded DNA (dsDNA) by single-stranded DNA (ssDNA) at 37 °C; the data were modelled as a second-order reaction approaching equilibrium, with a rate constant of 10 m-1 s-1 . We also followed the displacement from a DNA three-way junction (3WJ) by ssDNA. The presence of three internal mismatched bases in the middle of the invading strand did not prevent displacement from the 3WJ, but reduced the second-order rate constant by about 50 %. We attribute strand exchange in the dsDNA and 3WJ to a zero-toehold pathway from the blunt-ended duplex arms. The single-molecule approach demonstrated here will be useful for studying complex DNA networks.

In vitro isolation of small-molecule-binding aptamers with intrinsic dye-displacement functionality.

Aptamer-based sensors offer a powerful tool for molecular detection, but the practical implementation of these biosensors is hindered by costly and laborious sequence engineering and chemical modification procedures. We report a simple strategy for directly isolating signal-reporting aptamers in vitro through systematic evolution of ligands by exponential enrichment (SELEX) that transduce binding events into a detectable change of absorbance via target-induced displacement of a small-molecule dye. We first demonstrate that diethylthiatricarbocyanine (Cy7) can stack into DNA three-way junctions (TWJs) in a sequence-independent fashion, greatly altering the dye's absorbance spectrum. We then design a TWJ-containing structured library and isolate an aptamer against 3,4-methylenedioxypyrovalerone (MDPV), a synthetic cathinone that is an emerging drug of abuse. This aptamer intrinsically binds Cy7 within its TWJ domain, but MDPV efficiently displaces the dye, resulting in a change in absorbance within seconds. This assay is label-free, and detects nanomolar concentrations of MDPV. It also recognizes other synthetic cathinones, offering the potential to detect newly-emerging designer drugs, but does not detect structurally-similar non-cathinone compounds or common cutting agents. Moreover, we demonstrate that the Cy7-displacement colorimetric assay is more sensitive than a conventional strand-displacement fluorescence assay. We believe our strategy offers an effective generalized approach for the development of sensitive dye-displacement colorimetric assays for other small-molecule targets.

Logic circuit controlled multi-responsive branched DNA scaffolds.

A logic circuit controlled multi-responsive sensing platform built on a three-way DNA junction (TWJ) is reported. It enabled the construction of novel fluorescent sensing platforms responsive to any target out of HIV gene, ATP and pH value, and furthermore were logically regulated by two other targets and then behaved as different logic circuits, which consist of two tandem AND gates or cascaded NAND and INH gates by varying the positions of the fluorescent tags.

TWJ-Screen: an isothermal screening assay to assess ligand/DNA junction interactions in vitro.

The quest for chemicals able to operate at selected genomic loci in a spatiotemporally controlled manner is desirable to create manageable DNA damages. Mounting evidence now shows that alternative DNA structures, including G-quadruplexes and branched DNA (or DNA junctions), might hamper proper progression of replication fork, thus triggering DNA damages and genomic instability. Therefore, small molecules that stabilize these DNA structures are currently scrutinized as a promising way to create genomic defects that cannot be dealt with properly by cancer cells. While much emphasis has been recently given to G-quadruplexes and related ligands, we report herein on three-way DNA junctions (TWJ) and related ligands. We first highlight the biological implications of TWJ and their strategic relevance as triggers for replicative stress. Then, we describe a new in vitro high-throughput screening assay, TWJ-Screen, which allows for identifying TWJ ligands with both high affinity and selectivity for TWJ over other DNA structures (duplexes and quadruplexes), in a convenient and unbiased manner as demonstrated by the screening of a library of 25 compounds from different chemical families. TWJ-Screen thus represents a reliable mean to uncover molecular tools able to foster replicative stress through an innovative approach, thus providing new strategic opportunities to combat cancers.

Investigation of the Melting Behavior of DNA Three-Way Junctions in the Closed and Open States

Intramolecular three-way junctions are commonly found in both DNA and RNA. These structures are functionally relevant in ribozymes, riboswitches, rRNA, and during replication. In this work, we present a thermodynamic description of the unfolding of DNA intramolecular three-way junctions. We used a combination of spectroscopic and calorimetric techniques to investigate the folding/unfolding thermodynamics of two three-way junctions with a closed (Closed-J) or open (Open-J) junction and their appropriate control stem-loop motifs (GAAATT-Hp, CTATC-Hp, and Dumbbell). The overall results show that both junctions are stable over a wide range of salt concentrations. However, Open-J is more stable due to a higher enthalpy contribution from the formation of a higher number of basepair stacks whereas Closed-J has a defined structure and retains the basepair stacking of all three stems. The comparison of the experimental results of Closed-J and Open-J with those of their component stem-loop motifs allowed us to be more specific about their cooperative unfolding. For instance, Closed-J sacrifices thermal stability of the Dumbbell structure to maintain an overall folded state. At higher salt concentration, the simultaneous unfolding of the above domains is lost, resulting in the unfolding of the three separate stems. In contrast, the junction of Open-J in low salt retains the thermal and enthalpic stability of the Dumbbell structure although sacrificing stability of the CTATC stem. The relative stability of Dumbbell is the primary reason for the higher ΔG°(5), or free energy, value seen for Open-J at low salt. Higher salt not only maintains thermal stability of the Dumbbell structure in Open-J but causes the CTATC stem to fully fold.

Construction of Functional Biomaterials by Biomolecular Self-Assembly

Abstract This account discusses construction strategies for various functional biomaterials based on the designed self-assembly of biomolecules. Novel glycoclusters with regular intervals were developed by self-assembly of carbohydrate-conjugated oligodeoxyribonucleotides (ODNs) with the half-sliding complementary ODNs. Complexes of carbohydrate-modified DNA and lectin afforded a new regulation system for gene expression. DNA three-way junctions bearing self-complementary sticky-ends were self-assembled into nanometer-to-micrometer-sized spherical structures depending on the concentration. The three-way component design was extended to the design of an artificial trigonal peptide conjugate. The trigonal peptide conjugates bearing β-sheet-forming peptides or glutathione self-assembled into nano-sized spherical assemblies. Self-assembly of β-annulus peptide derived from tomato bushy stunt virus afforded artificial viral capsids, which can encapsulate and be modified with various molecules.

DNA Three-Way Junction for Differentiation of Single-Nucleotide Polymorphisms with Fluorescent Copper Nanoparticles.

A label- and enzyme-free fluorescent sensor for the detection of single-nucleotide polymorphisms (SNPs) at room temperature is proposed, using new copper nanoparticles (CuNPs) as fluorescent reporters. The CuNPs were constructed by using a DNA three-way junction (3WJ) template. In this assay, two complementary adenine/thymine-rich probes can hybridize with the wild-type target simultaneously to construct a 3WJ structure, serving as an efficient scaffold for the generation of CuNPs. However, the CuNPs produce weak fluorescence when the probes bind with a mutant-type target. SNPs can be identified by the difference in fluorescence intensity of the CuNPs. This SNPs detection strategy is straightforward, cost-effective, and avoids the complicated procedures of labeling or enzymatic reactions. The fluorescent sensor is versatile and can be applied to all types of mutation because the probes are programmable. Moreover, the sensor exhibits good detection performance in biological samples.

Formation of a DNA Mini-Dumbbell with a Quasi-Type II Loop.

Quasi-loops, wherein the backbone has a discontinuous site in the loop, have been found to provide structural flexibility in the formation of DNA three-way junctions. Recently, a highly compact mini-dumbbell (MDB) structure composed of two adjacent CCTG or TTTA type II loops has been reported. Yet, it remains elusive if the presence of a quasi-loop will also facilitate the formation of an MDB. In this study, the possibility of whether an MDB can be formed containing a quasi-type II loop has been investigated. We first demonstrate that two adjacent CTTG type II loops can also form an MDB, which is thermodynamically more stable than those formed by CCTG and TTTA loops. Then, we systematically introduce quasi-type II loops with their discontinuous sites at different backbone positions in the CTTG MDB. Our results show that an MDB can be formed with a quasi-type II loop, in which there is a discontinuous site between the third thymine and the fourth guanine loop residues. The possible inclusion of a quasi-loop in MDBs expands the sequence criteria for the formation of MDBs by natural DNA sequences.

Targeting Holliday junctions by origin DNA-binding protein of herpes simplex virus type 1

In the present paper, the interactions of the origin binding protein (OBP) of herpes simplex virus type 1 (HSV1) with synthetic four-way Holliday junctions (HJs) were studied using electrophoresis mobility shift assay and the FRET method and compared with the interactions of the protein with duplex and single-stranded DNAs. It has been found that OBP exhibits a strong preference for binding to four-way and three-way DNA junctions and possesses much lower affinities to duplex and single-stranded DNAs. The protein forms three types of complexes with HJs. It forms complexes I and II which are reminiscent of the tetramer and octamer complexes with four-way junction of HJ-specific protein RuvA of Escherichia coli. The binding approaches saturation level when two OBP dimers are bound per junction. In the presence of Mg2+ ions (≥2 mM) OBP also interacts with HJ in the stacked arm form (complex III). In the presence of 5 mM ATP and 10 mM Mg2+ ions OBP catalyzes processing of the HJ in which one of the annealed oligonucleotides has a 3′-terminal tail containing 20 unpaired thymine residues. The observed preference of OBP for binding to the four-way DNA junctions provides a basis for suggestion that OBP induces large DNA structural changes upon binding to Box I and Box II sites in OriS. These changes involve the bending and partial melting of the DNA at A+T-rich spacer and also include the formation of HJ containing Box I and Box II inverted repeats and flanking DNA sequences.

DNA charge transport: Moving beyond 1D

Charge transport across novel DNA junctions has been studied for several decades. From early attempts to move charge across DNA double crossover junctions to recent studies on DNA three-way junctions and G4 motifs, it is becoming clear that efficient cross-junction charge migration requires strong base-to-base electronic coupling at the junction, facilitated by favorable pi-stacking. We review recent progress toward the goal of manipulating and controlling charge transport through DNA junctions.

Branchpoint Structure of DNA Threeway Junctions

Branched structures of nucleic acids are widely observed in nature as intermediates during DNA repair, recombination and replication processes, as well as being a component of vital structures for protein synthesis. Using highresolution single-molecule Fo╮ster resonance energy transfer (SM-FRET), we showed recently that a DNA three way junction was not fully paired at the branchpoint, in spite of being fully complementary.(1) To investigate whether this expanded branchpoint was a general effect, or due to the specific sequence used, we have also studied a three-way junction with a GC-rich branchpoint. We report the results of SM-FRET and ensemble NMR experiments on this new junction, together with initial investigations of the branchpoint reactivity. 1. Sabir, Tara et al. Branchpoint Expansion in a Fully Complementary ThreeWay DNA Junction. J. Am. Chem. Soc. (2012) 134: 6280 6285