DNA Three-way Junction Research Articles

DNA three-way junction–ruthenium complex assemblies

Three-way junction building blocks were designed to construct novel 2D ruthenium–DNA assemblies. Discrete three-branched DNA motifs were formed with 1 to 3 sticky ends of 14-, 20- and/or 24-mer nucleotides. Hybridization with the complementary mono Ru–DNA conjugates afforded the formation of a family of three-way assemblies with 1 to 3 peripheral ruthenium complexes. The use of sticky ends of different lengths allowed us to modulate the number of metallic complexes introduced and also to extend the structure. Indeed, our construction strategy associated with the use of bis ruthenium–DNA conjugates permitted the assembly of double three-way junctions linked by DNA duplexes with or without ruthenium complexes in their centre.

Metal‐Locked DNA Three‐Way Junction

Metal‐Locked DNA Three‐Way Junction

Role of Specific Cations and Water Entropy on the Stability of Branched DNA Motif Structures

DNA three-way junctions (TWJs) are important intermediates in various cellular processes and are the simplest of a family of branched nucleic acids being considered as scaffolds for biomolecular nanotechnology. Branched nucleic acids are stabilized by divalent cations such as Mg(2+), presumably due to condensation and neutralization of the negatively charged DNA backbone. However, electrostatic screening effects point to more complex solvation dynamics and a large role of interfacial waters in thermodynamic stability. Here, we report extensive computer simulations in explicit water and salt on a model TWJ and use free energy calculations to quantify the role of ionic character and strength on stability. We find that enthalpic stabilization of the first and second hydration shells by Mg(2+) accounts for 1/3 and all of the free energy gain in 50% and pure MgCl(2) solutions, respectively. The more distorted DNA molecule is actually destabilized in pure MgCl(2) compared to pure NaCl. Notably, the first shell, interfacial waters have very low translational and rotational entropy (i.e., mobility) compared to the bulk, an entropic loss that is overcompensated by increased enthalpy from additional electrostatic interactions with Mg(2+). In contrast, the second hydration shell has anomalously high entropy as it is trapped between an immobile and bulklike layer. The nonmonotonic entropic signature and long-range perturbations of the hydration shells to Mg(2+) may have implications in the molecular recognition of these motifs. For example, we find that low salt stabilizes the parallel configuration of the three-way junction, whereas at normal salt we find antiparallel configurations deduced from the NMR. We use the 2PT analysis to follow the thermodynamics of this transition and find that the free energy barrier is dominated by entropic effects that result from the decreased surface area of the antiparallel form which has a smaller number of low entropy waters in the first monolayer.

The three-way junction DNAzyme based probe for label-free colorimetric detection of DNA

A novel three-way junction DNAzyme based probe has been designed for the colorimetric sensing of target DNA. Specifically, a DNAzyme-linked hairpin DNA is used as a signal probe. In the presence of target DNA, the signal probe, assistant probe and target DNA can hybridize with each other, resulting in the formation of a three-way junction DNA. At the same time, the signal probe is opened and the DNAzyme sequence in the signal probe is dehybridized. Subsequently, in the presence of hemin, the DNAzyme sequence forms a G-quadruplex–hemin complex, which catalyzes oxidation of 2, 2′-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) by H2O2 to the colored ABTS.−radical. The significant color changes can be distinguished visually. By the combination of the hairpin probe and the three-way junction DNA probe, the proposed sensor exhibits high recognition property for single-nucleotide polymorphisms (SNPs). This sensor allows the detection of target DNA at a concentration as low as 0.25nmol L−1. The proposed sensor is easy to fabricate, which avoids the tedious and expensive labeling procedures, and exhibits high selectivity against single-base mismatched DNA.

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.

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.

Expanding the rule set of DNA circuitry with associative toehold activation.

Toehold-mediated strand displacement has proven extremely powerful in programming enzyme-free DNA circuits and DNA nanomachines. To achieve multistep, autonomous, and complex behaviors, toeholds must be initially inactivated by hybridizing to inhibitor strands or domains and then relieved from inactivation in a programmed, timed manner. Although powerful and reasonably robust, this strategy has several drawbacks that limit the architecture of DNA circuits. For example, the combination between toeholds and branch migration (BM) domains is 'hard wired' during DNA synthesis thus cannot be created or changed during the execution of DNA circuits. To solve this problem, I propose a strategy called 'associative toehold activation', where the toeholds and BM domains are connected via hybridization of auxiliary domains during the execution of DNA circuits. Bulged thymidines that stabilize DNA three-way junctions substantially accelerate strand displacement reactions in this scheme, allowing fast strand displacement initiated by reversible toehold binding. To demonstrate the versatility of the scheme, I show (1) run-time combination of toeholds and BM domains, (2) run-time recombination of toeholds and BM domains, which results in a novel operation 'toehold switching', and (3) design of a simple conformational self-replicator.

Identifying three-way DNA junction-specific small-molecules

Three-way junction DNA (TWJ-DNA, also known as 3WJ-DNA) is an alternative secondary DNA structure comprised of three duplex-DNAs that converge towards a single point, termed the branch point. This point is characterized by unique geometrical properties that make its specific targeting by synthetic small-molecules possible. Such a targeting has already been demonstrated in the solid state but not thoroughly biophysically investigated in solution. Herein, a set of simple biophysical assays has been developed to identify TWJ-specific small-molecule ligands; these assays, inspired by the considerable body of work that has been reported to characterize the interactions between small-molecules and other higher-order DNA (notably quadruplex-DNA), have been calibrated with a known non-specific DNA binder (the porphyrin TMPyP4) and validated via the study of a small series of triazacyclononane (TACN) derivatives (metal-free or not) and the identification of a fairly-affinic and exquisitely TWJ-selective candidate (a TACN-quinoline construct named TACN-Q).

Small-Molecule-Triggered Manipulation of DNA Three-Way Junctions

DNA three-way junctions are frequently used in nanoarchitectures. Ligand-dependent designs that provide well-characterized building blocks for structure-switching DNA nanodevices are presented.

The Conformational Dynamics of Model Slipped Strand DNA Structures are Determined by Base Pairing in the Heteroduplex Arm

Slipped strand DNA is formed from the out-of-register pairing of direct repeats into thermodynamically stable hairpin structures. The formation of slipped strand DNA is hypothesized to be an important intermediate in triplet repeat expansion, a mechanism associated with many hereditary and anticipative neurodegenerative diseases. In this study, we use three-way DNA junctions as models for slipped strand DNA structures. Each of the three-way DNA molecules have two homoduplex arms and one heteroduplex arm, and the structures and conformational dynamics of the model substrates are characterized using single-molecule fluorescence resonance energy transfer (smFRET). FRET can measure changes in the relative distances between two fluorophores that are sufficiently close together (20-80 A). In our experiments, junctions are end-labeled with donor and acceptor fluorophores, and the efficiency of energy transfer E between the fluorophores is measured as a function of time to approximate the distances between the fluorophores and to monitor the conformational dynamics of the arms. We find that the junctions have trigonal pyramidal geometry. In addition, we observe that the junctions with two or more A-A mispairs on the heteroduplex arm transition between two conformational states with conformer transition rates that are dependent on ionic strength. This investigation sheds light on the structural and dynamical properties of junctions formed by slipped strand DNA which are important for understanding how enzymes recognize and process these structures.

A Yoctoliter-Scale DNA Reactor for Small-Molecule Evolution

The center of DNA three-way junctions, constituting a yoctoliter (10(-24) L) volume, is applied as an efficient reactor to create DNA-encoded libraries of chemical products. Amino acids and short peptides are linked to oligonucleotides via cleavable and noncleavable linkers. The oligonucleotide sequences contain two universal assembling domains at the center and a distal codon sequence specific for the attached building block. Stepwise self-assembly and chemical reactions of these conjugates in a combinatorial fashion create a library of pentapeptides in DNA three-way junctions in a single reaction vessel. We demonstrate the formation of an evenly distributed library of 100 peptides. Each library member contains a short synthetic peptide attached to a unique genetic code creating the necessary "genotype-phenotype" linkage essential to the process of in vitro molecular evolution. Selective enrichment of the [Leu]-enkephalin peptide from an original frequency of 1 in 10 million in a model library to a final frequency of 1.7% in only two rounds of affinity selection is described and demonstrates successful molecular evolution for a non-natural system.

CTAB-induced morphological transition of DNA micro-assembly from filled spheres to hollow capsules

DNA microspheres formed by self-assembly of DNA three-way junctions with self-complementary sticky-ends undergo an unexpected dynamic transformation from filled spheres into hollow capsules upon addition of cationic surfactants such as cetyltrimethylammonium bromide.

High-resolution cross-reactive array for alkaloids

A high-resolution cross-reactive array capable of classifying alkaloids over a range of concentrations was generated by systematic introduction of a nitroindole analog into a hydrophobic pocket within a DNA three-way junction to match structural motifs presented by the analytes.

Helix−Coil Transition of a Four-Way DNA Junction Observed by Multiple Fluorescence Parameters

The thermal denaturation of immobile four-way DNA ("Holliday-") junctions with 17 base pair arms was studied via fluorescence spectroscopic measurements. Two arms of the molecule were labeled at the 5'-end with fluorescein and tetramethylrhodamine, respectively. Melting was monitored by the fluorescence intensity of the dyes, the fluorescence anisotropy of tetramethylrhodamine, and Forster resonance energy transfer (FRET) between fluorescein and rhodamine. To fit the thermal denaturation curves of the four-way junctions, two basic thermodynamic models were tested: (1) all-or-none transitions assuming a molecularity of one, two, or four and (2) a statistical "zipper" model. The all-or-none models correspond to reaction mechanisms assuming that the cooperative melting unit (that is, the structure changing from complete helix to complete coil) consists of (1) one arm, (2) two neighboring arms (which have one continuous strand common to the two arms), or (3) all four arms. In each case, the melting of the cooperative unit takes place in a single step. The tetramolecular reaction model (four-arm melting) yielded unrealistically low van't Hoff enthalpy and entropy values, whereas the monomolecular model (one-arm melting) resulted in a poor fit to the experimental data. The all-or-none bimolecular (two neighboring arm model) fit gave intermediate standard enthalpy change (Delta H) values between those expected for the melting of a duplex with a total length between the helix lengths of one and two arms (17 and 34 base pairs). Simulations according to the zipper model fit the experimental curves best when the length of the simulated duplex was assumed to be 34 base pairs, the length of a single strand. This suggests that the most important parameter determining the melting behavior of the molecule is the end-to-end distance of the strands (34 bases) rather than the length of the individual arms (17 base pairs) and that the equilibrium concentration of partially denatured intermediate states has to be taken into account. These findings are in good agreement with results obtained for three-way DNA junctions ( Stuhmeier, F. ; Lilley, D. M. ; Clegg, R. M. Biochemistry 1997, 36, 13539 ). An interesting result is that the extent-of-melting curves derived from the fluorescence intensity and anisotropy nearly agree, whereas the curve derived from the FRET data shows a change prior to the melting. This may be an indication of a conformational change leaving the double-stranded structure intact but changing the end-to-end distance of the different arms in a way consistent with the transition to the extended square configuration ( Clegg, R. M. ; Murchie, A. I. ; Lilley, D. M. Biophys. J. 1994, 66, 99 ) of this branched molecule.

Molecular recognition and self-organization of three-way DNA junctions and supramolecular helicates

Molecular recognition and self-organization of three-way DNA junctions and supramolecular helicates

Nucleic acid secondary structures containing the double-headed nucleoside 5′(S)-C-(2-(thymin-1-yl)ethyl)thymidine

Oligodeoxynucleotides containing the double-headed nucleoside 5'(S)-C-(2-(thymin-1-yl)ethyl)thymidine were prepared by standard solid phase synthesis. The synthetic building block for incorporating the double-headed moiety was prepared from thymidine, which was stereoselectively converted to a protected 5'(S)-C-hydroxyethyl derivative and used to alkylate the additional thymine by a Mitsunobu reaction. The oligodeoxynucleotides were studied in different nucleic acid secondary structures: duplexes, bulged duplexes, three-way junctions and artificial DNA zipper motifs. The thermal stability of these complexes was studied, demonstrating an almost uniform thermal penalty of incorporating one double-headed nucleoside moiety into a duplex or a bulged duplex, comparable to the effects of the previously reported double-headed nucleoside 5'(S)-C-(thymin-1-yl)methylthymidine. The additional base showed only very small effects when incorporated into DNA or RNA three-way junctions. The various DNA zipper arrangements indicated that extending the linker from methylene to ethylene almost completely removed the selective minor groove base-base stacking interactions observed for the methylene linker in a (-3)-zipper, whereas interactions, although somewhat smaller, were observed for the ethylene linker in a (-4)-zipper motif.

Folding of 8-17 Deoxyribozyme Studied by Three-Color Alternating-Laser Excitation of Single Molecules

The folding of 8-17 deoxyribozyme was investigated by three-color alternating-laser excitation (3c-ALEX), a new single-molecule fluorescence resonance energy transfer (FRET) method we recently developed. Since 3c-ALEX has the capability of simultaneously sorting fluorescent molecules based on their labeling status and monitoring three interprobe distances of a biomolecule by employing three-color FRET, it is an ideal tool to study folding of multibranched molecules. The 8-17 deoxyribozyme, a DNA enzyme that cleaves a specific RNA substrate, is a good model system for a multibranched molecule, since it has the structure of a three-way DNA junction with a bulge. Labeling all three branches of the 8-17 with different fluorescent probes, we studied its [Mg2+]-dependent folding in a Na+ buffer solution. With the stoichiometric sorting capability of 3c-ALEX, we first selected only the triply labeled 8-17 in a solution of all heterogeneous mixtures and then simultaneously measured all three interprobe distances of the selected species. Our results show that the 8-17 folds into a pyramidal form upon increasing [Mg2+], in a similar way with [Zn2+] as found in an earlier study conducted at the ensemble level. The apparent dissociation constant of Mg2+ was more than 100 times larger than that of Zn2+ and showed considerable variance with buffer concentration. No clear sign of two-step folding was observed for Mg2+, in contrast to the case of Zn2+. Compared with the hammerhead ribozyme, the 8-17 was found to require 10 times higher [Mg2+] to undergo folding. By comparison with the folding of several inactive 8-17 analogues, we found that the two conserved sequences (A and G) of the triad loop of the shortest branch are critical elements for folding, especially for the folding at low [Mg2+]. Our results suggest that the role of the stem loop is to provide a scaffold for the two bases to be properly positioned for the necessary interaction and that the two bases are directly involved in the interaction that plays a critical role in folding. This work demonstrates that 3c-ALEX is a powerful single-molecule method to study the structure and folding of complex and multibranched biomolecules.

Structural basis for the recruitment of ERCC1-XPF to nucleotide excision repair complexes by XPA

The nucleotide excision repair (NER) pathway corrects DNA damage caused by sunlight, environmental mutagens and certain antitumor agents. This multistep DNA repair reaction operates by the sequential assembly of protein factors at sites of DNA damage. The efficient recognition of DNA damage and its repair are orchestrated by specific protein-protein and protein-DNA interactions within NER complexes. We have investigated an essential protein-protein interaction of the NER pathway, the binding of the XPA protein to the ERCC1 subunit of the repair endonuclease ERCC1-XPF. The structure of ERCC1 in complex with an XPA peptide shows that only a small region of XPA interacts with ERCC1 to form a stable complex exhibiting submicromolar binding affinity. However, this XPA peptide is a potent inhibitor of NER activity in a cell-free assay, blocking the excision of a cisplatin adduct from DNA. The structure of the peptide inhibitor bound to its target site reveals a binding interface that is amenable to the development of small molecule peptidomimetics that could be used to modulate NER repair activities in vivo.

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.