Triplex-forming oligonucleotides: a third strand for DNA nanotechnology

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.

Figure 1.

The double-helix as a secondary structural element: Structural motifs assembled through single (A), double (B), and multiple (C–E) strand crossovers between adjacent double-helical domains. The size of these structures can be extended by the appropriate positioning of complementary single-stranded overhangs (sticky-ends): (B) 2D array generated from ca. 10⁶ copies of a double-crossover molecule; (D) tetrahedron assembled from four copies of a three-point star motif; and (E) 3D crystal formed from ca. 10¹² copies of a tensegrity triangle motif. The single-layer structures shown in (B) and (C) were imaged by atomic force microscopy; the tetrahedral structure shown in (D) was reconstructed from cryo-electron microscopy analysis; whilst the crystal shown in (E) was imaged by light microscopy and its underlying DNA structure later solved by X-ray diffraction analysis. The zig-zag lines in (A) represent half-helical turns and arrows reflect the 5′-3′ polarity of strands. Where possible non-crossover strands are shown in gold and cylinders denote the double-helical regions within each motif. Adapted from (4,9,14,15) with permission.

Figure 2.

The parallel triple-helix: (A) NMR structure of a parallel triplex formed by the binding of a third strand within the major groove of a polypurine-polypyrimdine duplex (PDB code: 13DX). (B) Chemical structures of parallel T-AT and C⁺-GC triplets. The notation X-RY refers to a triplet in which the third strand base (X) binds to a purine (R) and pyrimidine (Y) base pair of its target duplex. (C) Sequence of a typical 13-mer triplex that we have used in our own work, also shown in a zig-zag format. The third strand (X-strand) is shown in dark blue and the duplex oligopurine (R-strand) and oligopyrimidine (Y-strand) strands in orange and grey, respectively. Where possible zig-zag diagrams, strand colourings and X, R and Y strand labels remain constant throughout the text.

Figure 3.

Triplex-based devices that respond to pH change: (A) (i) pH-dependent device based on molecular tweezers. The complex contains an appropriately positioned FRET pair (F1 and F2) that allows the opening and closure of the device to be monitored upon pH change; (ii) FRET data generated through the repeated pH cycling of such a device. Adapted from (57) with permission. (B) (i) pH-dependent device based on a simple intramolecular triplex. The complex contains an appropriately positioned fluorophore (F) and quencher (Q) that allows the opening and closure of the device to be monitored upon pH change. (ii) Varying the relative T-AT and C⁺-GC content of such a device allows the sensing of different pH values. Adapted from (60) with permission. (C) (i) pH-dependent complex composed of an intramolecular triplex and graphene oxide (GO) used to monitor pH changes associated with apoptosis in living cells. At high pH, the triplex-forming sequence interacts with GO, whilst at low pH triplex formation prevents this interaction. Since GO quenches fluorescence a fluorophore attached to the triplex allows association, and hence the pH of the solution, to be monitored directly. (ii) Fluorescence images of living cells transfected with the GO-device. Adapted from (61) with permission.

Figure 4.

Triplex-directed chemical reactions: (A) Directing amide bond formation by triplex formation. A carboxylic acid group attached to the triplex-forming sequence is positioned adjacent to a terminal (amine 1) or central amine (amine 2) upon duplex and triplex formation, respectively. The reaction is initiated by the addition of a condensation agent. Adapted from (62) with permission. (B) Control of copper-catalysed alkyne-azide cycloaddition reactions. (i) Upon duplex formation the reaction leads to the linkage of the two duplex strands (reaction 1). (ii) Subsequent addition of a triplex binder, which promotes triplex formation, leads to the linkage of the third strand to the pre-linked duplex (reaction 2). Adapted from (64) with permission.

Figure 5.

Triplex-based devices capable of capturing and releasing molecules: (A) Control of a tweezer-like DX device that captures/releases single-stranded DNA. The captured strand binds through triplex formation and is subsequently held in place by the addition of toe-hold containing strand that forms the second crossover of the molecule. Upon increasing the pH, the oligonucleotide remains trapped, and is only released by removal of the toe-hold containing strand by addition of its W–C complement. Adapted from (65) with permission. (B) Control of a clamp-like device that detects ATP. A triplex generated within the molecule by the addition of its R-strand brings into close proximity two halves of a split aptamer capable of binding ATP. Adapted from (67) with permission.

Figure 6.

Triplex-mediated strand displacement reactions: (A) (i) Mechanism of third strand displacement by addition of its W–C complement; (ii) the addition of a toehold to the end of the third strand can be used to enhance the reaction. (B) Mechanism of duplex strand displacement using a short triplex domain as a toehold. Third strand binding positions strand S2 adjacent to identical strand S and as a result leads to its gradual displacement from the duplex by branch migration. Adapted from (70) with permission. (C) (i) OH⁻-activated duplex strand displacement. At low pH, binding of a third strand blocks access of the invading strand to the duplex toehold and prevents strand displacement, whilst at high pH the third strand dissociates and the displacement reaction can proceed. (ii) H⁺-activated duplex strand displacement. At low pH triplex formation with a clamp-like oligonucleotide containing both Y2- and X-strands leads to the displacement of identical strand Y. Adapted from (71) with permission.

Figure 7.

Hierarchical formation of extended DNA complexes through the control of strand displacement reactions. (A) Formation of a DNA concatemer at basic pH. The system is composed of two metastable hairpin species that react with each other in the presence of an initiator strand. The initiator binds to a toehold region on the hairpin of one species (HP1) and through strand displacement exposes a new single-stranded region that opens the hairpin of the second species (HP2), generating a concatemer of the two duplexes. (i) At low pH the binding of the initiator to HP1 is inhibited due to the formation of a triplex which sequesters the toehold portion of the molecule. (ii) Increasing the pH leads to the dissociation of the third strand, which allows initiator binding, and subsequent polymerisation of the two hairpin species. Adapted from (72) with permission. (B) Formation of double-crossover lattices at basic pH. The system is composed of a triplex-based strand displacement circuit that activates a downstream self-assembling reaction: the formation of a DX array similar in design to the one shown in Figure 1B. (i) The circuit is initiated by the binding of a catalyst strand to a pH-dependent substrate. At low pH, triplex formation prevents this interaction, whilst at high pH the reaction can proceed. (ii) Catalyst binding releases a deprotector strand through strand displacement. (ii) The deprotector strand then associates with a protected tile that is made reactive through the displacement of protecting strands that cover the sticky-ends of the molecule, resulting in array formation. Adapted from (73) with permission.

Figure 8.

Hierarchical assembly and/or dissociation of extended DNA complexes through pH change: (A) Triplex motif used for reconfiguring the interactions of hexagonal origami tiles. At high pH, the strands form two inter-linked duplexes between tile 1 and tile 2, whilst at low pH one strand of one of the duplex partners folds back and forms an intramolecular triplex leading to the dissociation of the two tiles. Adapted from (74) with permission. (B) Triplex motif used for reconfiguring the interaction of a three-point star motif into a DNA tetrahedron similar to the one shown in Figure 1D. At low pH, the two triplex-modified sticky-ends interact, whilst at high pH they do not. Adapted from (75) with permission.

Figure 9.

Hierarchical assembly/dissociation of heterogeneous complexes: (A) pH-dependent aggregation of gold nanoparticles. One set of gold nanoparticles is functionalized with a third strand, whilst the second set is functionalized with its duplex partner. Dissociation/assembly of the two sets of gold particles into a 3D network is controlled through pH change. Adapted from (76) with permission. (B) Small-molecule induced aggregation of single-walled carbon nanotubes. Each nanotube is functionalised with one or more polyT-polyA duplexes capable of repartitioning into a polyT-polyA-polyT triplex upon addition of a triplex-stabilizing ligand, such as coralyne. Adapted from (84) with permission; (C) pH-responsive hydrogels. The acrylamide chains are functionalized with a single DNA strand capable of forming either an intermolecular duplex at high pH, or intramolecular triplex at low pH leading to the association and dissociation of the copolymer chains, respectively. Adapted from (85) with permission.

Figure 10.

Triplex-directed targeting of DNA nanostructures: Triplex sequence addressability of various DNA architectures. (A) Targeting of an individual sequence within a hexagonal array generated from three-way branched oligonucleotides. The complex contains an appropriately positioned FRET pair (F1 and F2) that allows the association and dissociation of the TFO to be monitored by pH change. Adapted from (90) with permission. (B) Triplex-directed scaffolding of a double-crossover tile and array with a streptavidin proteins. Adapted from (91) with permission. (C) Targeting of an individual sequence positioned within a DNA origami frame. Binding of the TFO results in the association of the two duplexes running along the centre of the frame into an X-shaped structure that is visualised by AFM. Adapted from (93) with permission. (D) Triplex- directed scaffolding of a tensegrity triangle crystal with a cyanine dye. Adapted from (94) with permission.

Figure 11.

Triplex-directed modification of DNA nanostructures: (A) triplex-directed intercalation and photo-cross-linking of a tethered psoralen molecule to a TpA step introduced adjacent to a TFO target sequence. UV exposure results in a 2+2 cycloaddition reaction with the adjacent thymidines (shown in light blue), thereby cross-linking the two strands. (B) Appropriate targeting of a TpA step and target sequence embedded between adjacent tiles of a tensegrity triangle crystal allows cross-linking across the sticky-ends and increases crystal stability. Adapted from (95) with permission.