Abstract
DNA self-assembly has proved to be a useful bottom-up strategy for the construction of user-defined nanoscale objects, lattices and devices. The design of these structures has largely relied ...on exploiting simple base pairing rules and the formation of double-helical domains as secondary structural elements. However, other helical forms involving specific non-canonical base-base interactions have introduced a novel paradigm into the process of engineering with DNA. The most notable of these is a three-stranded complex generated by the binding of a third strand within the duplex major groove, generating a triple-helical ('triplex') structure. The sequence, structural and assembly requirements that differentiate triplexes from their duplex counterparts has allowed the design of nanostructures for both dynamic and/or structural purposes, as well as a means to target non-nucleic acid components to precise locations within a nanostructure scaffold. Here, we review the properties of triplexes that have proved useful in the engineering of DNA nanostructures, with an emphasis on applications that hitherto have not been possible by duplex formation alone.
Abstract
The sequence-specific recognition of duplex DNA by unmodified parallel triplex-forming oligonucleotides is restricted to low pH conditions due to a necessity for cytosine protonation in the ...third strand. This has severely restricted their use as gene-targeting agents, as well as for the detection and/or functionalisation of synthetic or genomic DNA. Here I report that the nucleobase 6-amino-5-nitropyridin-2-one (Z) finally overcomes this constraint by acting as an uncharged mimic of protonated cytosine. Synthetic TFOs containing the nucleobase enabled stable and selective triplex formation at oligopurine-oligopyrimidine sequences containing multiple isolated or contiguous GC base pairs at neutral pH and above. Moreover, I demonstrate a universal strategy for the enzymatic assembly of Z-containing TFOs using its commercially available deoxyribonucleotide triphosphate. These findings seek to improve not only the recognition properties of TFOs but also the cost and/or expertise associated with their chemical syntheses.
Per- and polyfluoroalkyl substances (PFAS) make up a large group of persistent anthropogenic chemicals which are difficult to degrade and/or destroy. PFAS are an emerging class of contaminants, but ...little is known about the long-term health effects related to exposure. In addition, technologies to identify levels of contamination in the environment and to remediate contaminated sites are currently inadequate. In this opinion-type discussion paper, a team of researchers from the University of Connecticut and the University at Albany discuss the scientific challenges in their specific but intertwined PFAS research areas, including rapid and low-cost detection, energy-saving remediation, the role of T helper cells in immunotoxicity, and the biochemical and molecular effects of PFAS among community residents with measurable PFAS concentrations. Potential research directions that may be employed to address those challenges and improve the understanding of sensing, remediation, exposure to, and health effects of PFAS are then presented. We hope our account of emerging problems related to PFAS contamination will encourage a broad range of scientific experts to bring these research initiatives addressing PFAS into play on a national scale.
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•PFAS receive extensive attention as a class of emerging contaminants.•Exposure, health effects, sensing, and remediation of PFAS are focused on.•Scientific challenges for emerging PFAS contaminants are discussed.•Potential research directions to address those challenges are presented.
Here, we present a cross-linking approach to covalently functionalize and stabilize DNA origami structures in a one-pot reaction. Our strategy involves adding nucleotide sequences to adjacent staple ...strands, so that, upon assembly of the origami structure, the extensions form short hairpin duplexes targetable by psoralen-labeled triplex-forming oligonucleotides bearing other functional groups (pso-TFOs). Subsequent irradiation with UVA light generates psoralen adducts with one or both hairpin staples leading to site-specific attachment of the pso-TFO (and attached group) to the origami with ca. 80% efficiency. Bis-adduct formation between strands in proximal hairpins further tethers the TFO to the structure and generates “superstaples” that improve the structural integrity of the functionalized complex. We show that directing cross-linking to regions outside of the origami core dramatically reduces sensitivity of the structures to thermal denaturation and disassembly by T7 RNA polymerase. We also show that the underlying duplex regions of the origami core are digested by DNase I and thus remain accessible to read-out by DNA-binding proteins. Our strategy is scalable and cost-effective, as it works with existing DNA origami structures, does not require scaffold redesign, and can be achieved with just one psoralen-modified oligonucleotide.
Reconfigurable structures engineered through DNA hybridization and self-assembly offer both structural and dynamic applications in nanotechnology. Here, we have demonstrated that strand displacement ...of triplex-forming oligonucleotides (TFOs) can be translated to a robust macroscopic DNA crystal by coloring the crystals with covalently attached fluorescent dyes. We show that three different types of triplex strand displacement are feasible within the DNA crystals and the bound TFOs can be removed and/or replaced by (a) changing the pH from 5 to 7, (b) the addition of the Watson–Crick complement to a TFO containing a short toehold, and (c) the addition of a longer TFO that uses the duplex edge as a toehold. We have also proved by X-ray diffraction that the structure of the crystals remains as designed in the presence of the TFOs.
An orthogonal, noncovalent approach to direct the assembly of higher-order DNA origami nanostructures is described. By incorporating perfluorinated tags into the edges of DNA origami tiles we control ...their hierarchical assembly via fluorous-directed recognition. When we combine this approach with Watson–Crick base-pairing we form discrete dimeric constructs in significantly higher yield (8x) than when either molecular recognition method is used in isolation. This integrated “catch-and-latch” approach, which combines the strength and mobility of the fluorous effect with the specificity of base-pairing, provides an additional toolset for DNA nanotechnology, one that enables increased assembly efficiency while requiring significantly fewer DNA sequences. As a result, our integration of fluorous-directed assembly into origami systems represents a cheap, atom-efficient means to produce discrete superstructures.
DNA is a very useful molecule for the programmed self‐assembly of 2D and 3D nanoscale objects.1 The design of these structures exploits Watson–Crick hybridization and strand exchange to stitch linear ...duplexes into finite assemblies.2–4 The dimensions of these complexes can be increased by over five orders of magnitude through self‐assembly of cohesive single‐stranded segments (sticky ends).5, 6 Methods that exploit the sequence addressability of DNA nanostructures will enable the programmable positioning of components in 2D and 3D space, offering applications such as the organization of nanoelectronics,7 the direction of biological cascades,8 and the structure determination of periodically positioned molecules by X‐ray diffraction.9 To this end we present a macroscopic 3D crystal based on the 3‐fold rotationally symmetric tensegrity triangle3, 6 that can be functionalized by a triplex‐forming oligonucleotide on each of its helical edges.
Not so crystal clear: A macroscopic DNA crystal is presented based on the 3‐fold symmetrical tensegrity triangle that has been functionalized with a triplex‐forming oligonucleotide at each of its double‐helical edges. Attachment of a fluorescent dye to the oligonucleotide led to its incorporation within the asymmetric unit cell of the crystal and yielded colored DNA crystals.
We have achieved recognition of all 4 bp by triple helix formation at physiological pH, using triplex-forming oligonucleotides that contain four different synthetic nucleotides. BAU ...2′-aminoethoxy-5-(3-aminoprop-1-ynyl)uridine recognizes AT base pairs with high affinity, MeP (3-methyl-2 aminopyridine) binds to GC at higher pHs than cytosine, while APP (6-(3-aminopropyl)-7-methyl-3H-pyrrolo2,3-dpyrimidin-2(7H)-one) and S N-(4-(3-acetamidophenyl)thiazol-2-yl-acetamide) bind to CG and TA base pairs, respectively. Fluorescence melting and DNase I footprinting demonstrate successful triplex formation at a 19mer oligopurine sequence that contains two CG and two TA interruptions. The complexes are pH dependent, but are still stable at pH 7.0. BAU, MeP and APP retain considerable selectivity, and single base pair changes opposite these residues cause a large reduction in affinity. In contrast, S is less selective and tolerates CG pairs as well as TA.
The tensegrity triangle is a robust DNA motif that can self-assemble to generate macroscopic three-dimensional crystals. However, the stability of these crystals is dependent on the high ionic ...conditions used for crystal growth. Here we demonstrate that a triplex-forming oligonucleotide can be used to direct the specific intercalation, and subsequent photo-cross-linking, of 4,5',8-trimethylpsoralen to single or multiple loci within or between the tiles of the crystal. Cross-linking between the tiles of the crystal improves their thermal stability. Such an approach is likely to facilitate the removal of crystals from their mother liquor and may prove useful for applications that require greater crystal stability.
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•All the edges of a DNA tetrahedron melt at the same temperature.•Hoechst 33258 stabilises a DNA tetrahedron by binding to its AATT recognition sequence.•Stabilisation propagates ...throughout the tetrahedron which melts as a single entity.
DNA strands can be designed to assemble into stable three-dimensional structures, based on Watson-Crick base pairing rules. The simplest of these is the DNA tetrahedron that is composed of four oligonucleotides. We have re-designed the sequence of a DNA tetrahedron so that it contains a single (AATT) binding site for the minor groove binding ligand Hoechst 33258. We examined the stability of this structure by placing fluorescent groups within each of its edges and have shown that all the edges melt at the same temperature in the absence of the ligand. The minor groove ligand still binds to its recognition sequence within the tetrahedron and increases the melting temperature of the folded complex. This ligand-induced stabilisation is propagated into the adjacent helical arms and the tetrahedron melts as a single entity in a cooperative fashion.
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