Recognition of DNA three-way junctions by metallosupramolecular cylinders: gel electrophoresis studies

The interaction of metallosupramolecular cylinders with DNA three-way junctions has been studied by gel electrophoresis. A recent X-ray crystal structure of a palindromic oligonucleotide forming part of a complex with such a cylinder revealed binding at the heart of a three-way junction structure. The studies reported herein confirm that this is not solely an artefact of crystallisation and reveal that this is a potentially very powerful new mode of DNA recognition with wide scope. The cylinders are much more effective at stabilizing three-way junctions than simple magnesium di-cations or organic or metallo-organic tetra-cations, with the M cylinder enantiomer being more effective than P. The recognition is not restricted to three-way junctions formed from palindromic DNA with a central AT step at the junction; non-palindromic three-way junctions and those with GC steps are also stabilised. The cylinder is also revealed to stabilise other Y-shaped junctions, such as that formed at a fraying point in duplex DNA (for example, a replication fork), and other DNA three-way junction structures, such as those containing unpaired nucleotides, perhaps by opening up this structure to access the central cavity.     

Cross-reactive arrays based on three-way junctions

We report herein a novel system for the parallel processing of molecular recognition events utilizing arrays of oligonucleotide-based fluorescent sensors to characterize hydrophobic molecules in solution. The binding domains of the sensors were based on three-way junctions that utilize double helical stems as framework regions to reliably fold regardless of variations in or around the binding domain. A reporting domain was introduced by the specific substitution of a single phosphodiester group with a phosphorothioate, followed by selective functionalization with a fluorophore. The sensors were organized into cross-reactive arrays to yield characteristic fingerprints for samples containing hydrophobic molecules. The fingerprints can be used to characterize steroids in solution, including complex biologically important fluids. Arrays have the potential for clinical applications such as the detection of gross errors in steroidogenesis.     

The electrophoretic mobility of DNA three-way junctions is affected by the sequence of overhanging single-stranded ends

The folding of three- and four-way DNA junctions is often assessed by comparing the electrophoretic mobility of restriction enzyme fragments, using the long-short arm assay. We have compared the mobility of synthetic three-way junctions that contain identical branch point sequences, but different restriction sites in the arms. We show that the mobility of fragments is affected by the sequence of the overhanging ends. In general, GC-rich overhangs produce fragments with anomalous mobilities. These anomalies can be prevented by treating the cleaved junctions with mung bean endonuclease, elevating the electrophoresis temperature or using blunt cleaving restriction endonucleases.     

Artificial peptide-nanospheres self-assembled from three-way junctions of beta-sheet-forming peptides

Rational design of self-assembly of proteins, which plays pivotal roles in biology, is an important subject for biotechnology and also bottom-up nanotechnology. This paper has proposed a novel strategy for construction of artificial peptide-nanospheres by self-assembly. Mimicking formation of spherical viruses and clathrin, we designed a novel C3-symmetric peptide conjugate bearing three beta-sheet-forming peptides. These peptide conjugates formed antiparallel beta-structures and self-assembled into nanospheres with the size of about 20 nm in the acidic solution.     

DNA loops and semicatenated DNA junctions

Background  

Alternative DNA conformations are of particular interest as potential signals to mark important sites on the genome. The structural variability of CA microsatellites is particularly pronounced; these are repetitive poly(CA) · poly(TG) DNA sequences spread in all eukaryotic genomes as tracts of up to 60 base pairs long. Many in vitro studies have shown that the structure of poly(CA) · poly(TG) can vary markedly from the classical right handed DNA double helix and adopt diverse alternative conformations. Here we have studied the mechanism of formation and the structure of an alternative DNA structure, named Form X, which was observed previously by polyacrylamide gel electrophoresis of DNA fragments containing a tract of the CA microsatellite poly(CA) · poly(TG) but had not yet been characterized.     

Branchpoint expansion in a fully complementary three-way DNA junction

Branched nucleic acid molecules serve as key intermediates in DNA replication, recombination, and repair; architectural elements in RNA; and building blocks and functional components for nanoscience applications. Using a combination of high-resolution single-molecule FRET, time-resolved spectroscopy, and molecular modeling, we have probed the local and global structure of a DNA three-way junction (3WJ) in solution. We found that it adopts a Y-shaped, pyramidal structure, in which the bases adjacent to the branchpoint are unpaired, despite the full Watson-Crick complementarity of the molecule. The unpairing allows a nanoscale cavity to form at the junction center. Our structure accounts for earlier observations made of the structure, flexibility, and reactivity of 3WJs. We anticipate that these results will guide the development of new DNA-based supramolecular receptors and nanosystems.     

Gel electrophoretic analysis of DNA branched junctions

Gel electrophoresis has provided much of the detailed information we have about the properties of DNA junctions, stable branched molecules formed from oligonucleotide or polynucleotide strands. Here we review these applications, and present the results of an electrophoretic investigation of conformationally restricted junctions formed by covalently connecting two different pairs of strands in a junction with four arms. Native gel electrophoresis is employed to establish the formation and stoichiometry of the multistrand complexes. Ferguson analysis of native gel mobility shows that junctions have retardation coefficients that are distinct from those of linear DNA duplexes. Denaturing gel electrophoresis is the primary tool for characterizing junctions that have been covalently linked together to form both linear and macrocyclic oligomers of junctions (oligojunctions). Radioactively labelled strands enable one to monitor the progress of the ligation reaction: both linear and closed cyclic molecules result, and these can be distinguished by applying Ferguson analysis to denaturing gels. Combinations of exonuclease III, restriction enzymes and sequencing reactions have been applied to oligojunction molecules, and the results are all analyzed on denaturing gels. Junctions containing intramolecular "tethers" that restrict the conformation freedom of the complex comprise a new system for analyzing the conformations of branched molecules. In these tethered junctions, the ability of arms to move relative to each other is restricted substantially by covalently connecting pairs of arms in the original complex with short, flexible loops. The two tethers used here constrain the helical domains of the structure to be roughly parallel or anti-parallel. In this article, we use Ferguson analysis to compare two tethered junctions with an untethered junction. At high gel concentrations, the mobility of the untethered complex is found to be closer to that of the molecule tethered anti-parallel than to the one tethered parallel. Curvature in the Ferguson plots for all three of these junctions is detected over a range of compositions. At low gel concentrations, differences in electrophoretic mobility persist, suggesting that the untethered junction differs in charge as well as conformational freedom from the tethered analogs. We expect that studies of this kind will be able to define the conformational repertoire of junctions of different kinds, and to explore the effects of electrophoresis on these states.     

Artificial DNA cutters for DNA manipulation and genome engineering

This tutorial review provides recent developments in artificial cutters for site-selective scission of DNA with the focus on chemistry-based DNA cutters. They are useful tools for molecular biology and biotechnology, since their site-selectivity of scission is much higher than that of naturally occurring restriction enzymes and also their scission site is freely chosen. In order to prepare these cutters, a DNA-cutting molecule is combined with a sequence-recognizing molecule in a covalent or non-covalent way. At targeted sites in single-stranded and double-stranded DNAs, the scission occurs via either oxidative cleavage of nucleotides or hydrolysis of phosphodiester linkages. Among many successful examples, an artificial restriction DNA cutter, prepared from Ce(iv)/EDTA and pseudo-complementary peptide nucleic acid, hydrolyzed double-stranded DNA at the target site. The scission site and scission specificity are determined simply in terms of the Watson-Crick rule so that even the whole genome of human beings was selectively cut at one predetermined site. Consistently, homologous recombination in human cells was successfully promoted by this tool. For the purpose of comparison, protein-based DNA cutters (e.g., zinc finger nucleases) are also briefly described. The potential applications of these cutters and their future aspects are discussed.     

The DNA three-way junction as a mould for tripartite chromophore assembly

The DNA three-way junction serves as a scaffold for the molecular organization of non-nucleosidic alkynylpyrene and perylenediimide chromophores located at the branch point of the structure. Depending on the composition of the tripartite assembly, the constructs possess distinct spectroscopic properties, ranging from monomer or excimer fluorescence to completely quenched tripartite aggregates.     

Electrochemical DNA three-way junction based sensor for distinguishing chiral metallo-supramolecular complexes

Here we report a signal-on three-way junction based-electrochemical DNA sensor for distinguishing chiral metallo-supramolecular complexes with an enantioselective recognition ratio of about 3.5. This platform is easy to fabricate, simple to operate and readily regenerated.     

Evolution of high-branching deoxyribozymes from a catalytic DNA with a three-way junction

Here, we report the evolution of two star-shaped (five-way junction) deoxyribozymes from a catalytic DNA containing a three-way junction scaffold. The transition was shown to be a switch rather than a gradual progression. The star-shaped motifs, surprisingly, only took five selection cycles to be detected, and another four to dominate the evolving population. Chemical probing experiments indicated that the two deoxyribozymes belong to the same family despite noticeable variations in both the primary sequence and the secondary structure. Our findings not only describe the evolution of high-branching nucleic acid structures from a low-branching catalytic module, but they also illustrate the idea of deriving a rare structural motif by sampling the sequence variants of a given functional nucleic acid.     

Tensegrity: construction of rigid DNA triangles with flexible four-arm DNA junctions

A tensegrity strategy has been explored to construct a rigid geometrical structure (triangles) from flexible DNA four-arm junctions. The resulting DNA triangles could self-assemble into 1D and 2D arrays. This tensegrity strategy is expected to play an important role in the design of biomimetic nanomaterials.     

Ultrasensitive assay of ctDNA based on DNA triangular prism and three-way junction nanostructures

Circulating tumor DNA(ctDNA) refers to a class of acellular nucleic acids carrying genetic features of primary tumor,which can be regarded as a promising noninvasive biomarker for cancer diagnosis.The development of ctDNA assay is an important component of liquid biopsy.In this study,we have fabricated a novel electrochemical strategy for ultrasensitive detection of ctDNA combining the merits of strand displacement amplification and DNA nanostructures.Stable DNA triangular prism is firstly selfassembled and modified on the electrode surface.After target initiated strand displacement polymerization reaction,the generated DNA product helps the formation of three-way junction nanostructure on triangular prism,which localizes electrochemical species.By carefully investigating the electrochemical responses,the limit of detection(LOD) for ctDNA assay as low as 48 amol/L is achieved.This proposed electrochemical biosensor shows great potential for clinical applications.     

Programmable one-pot multistep organic synthesis using DNA junctions

A system for multistep DNA-templated synthesis is controlled by the sequential formation of DNA junctions. Reactants are attached to DNA adapters which are brought together by hybridization to DNA template strands. This process can be repeated to allow sequence-controlled oligomer synthesis while maintaining a constant reaction environment, independent of oligomer length, at each reaction step. Synthesis can take place in a single pot containing all required reactive monomers. Different oligomers can be synthesized in parallel in the same vessel, and the products of parallel synthesis can be ligated, reducing the number of reaction steps required to produce an oligomer of a given length.     

Bio-switchable optofluidic lasers based on DNA Holliday junctions

Bio-switchable optofluidic lasers based on DNA Holliday junctions were demonstrated. Nearly 100% wavelength switching was achieved through reversible conformational change of the Holliday junction controlled by magnesium ionic strength.     

Electrophoretic manipulation of single DNA molecules in nanofabricated capillaries

We demonstrate the use of nanofabricated capillaries, integrated as part of a microfluidic structure, to study the electrophoretic behaviour of single, fluorescently-labelled, molecules of DNA as a function of capillary size. The nanocapillaries, fabricated using a focused ion beam, have cross-sections down to 150 x 180 nm. Control of single-molecule direction and velocity was achieved using voltage manipulation. DNA mobility was found to increase with decreasing cross-section, which we interpret in terms of reduced electro-osmotic counter-flow. Such nanofabricated capillaries as part of larger fluidic structures have great potential for biotechnology, particularly single molecule manipulation and analysis.     

A cost-effective detection of low-abundance mutation with DNA three-way junction structure and lambda exonuclease

We presented a low-abundance mutation detection method with lambda exonuclease and DNA threeway junction structure.The assistant strand in the DNA three-way junction structure could regulate the reaction system from the kinetics and thermodyna mics aspects.The optimization of the assista nt strand helps to improve the selectivity of the mutant-type DNA to the wild-type DNA about 35 times.Moreover,the cost of the optimization process could be saved by about 90%.The method was applied to the detection of a human ovarian cancer-related gene mutation BRCA1(rs1799949,c.2082 CT).The limit of detection to the mutation abundance in the DNA three-way junction structure system(0.2%) was one order lower compared with that in the double-stranded DNA structure system(2%).The mutation abundance in different standard samples was quantitively measured,and the results were consistent with the initial abundance in the standard samples.     

Highly sensitive electrochemical detection of mercury (II) via single ion-induced three-way junction of DNA

A “signal-on” electrochemical sensing strategy was designed for highly sensitive and selective detection of mercury (II) via its induction to three-way junction of DNA (DNA-TWJ). The TWJ consisted of the capture probe that was self-assembled on a gold electrode surface through SAu bond, the signal probe that was labeled with ferrocene (Fc) and contained single T–T mismatch to capture probe, and an assistant probe for the formation of DNA-TWJ upon the presence of mercury (II). This process caused the Fc tag approaching the electrode for fast electron transfer and thus increased the oxidation current. The “signal-on” sensing method could detect Hg^2 + ranging from 0.005 to 100 nM. The assay was simple and fast. It showed potential application in on-site and real-time Hg^2 + detection.     

Ultrafast reversible photo-cross-linking reaction: toward in situ DNA manipulation

We describe a novel ultrafast reversible DNA interstrand photo-cross-linking reaction via 3-cyanovinylcarbazole nucleoside ( (CNV)K). Oligodeoxynucleotide (ODN) containing (CNV)K can be photo-cross-linked by irradiation at 366 nm for 1 s, and the photo-cross-linked ODN can be split by irradiation at 312 nm for 60 s.