Three's a crowd - stabilisation, structure, and applications of DNA triplexes

Abstract

DNA is a strikingly flexible molecule and can form a variety of secondary structures, including the triple helix, which is the subject of this review. The DNA triplex may be formed naturally, during homologous recombination, or can be formed by the introduction of a synthetic triplex forming oligonucleotide (TFO) to a DNA duplex. As the TFO will bind to the duplex with sequence specificity, there is significant interest in developing TFOs with potential therapeutic applications, including using TFOs as a delivery mechanism for compounds able to modify or damage DNA. However, to combine triplexes with functionalised compounds, a full understanding of triplex structure and chemical modification strategies, which may increase triplex stability or in vivo degradation, is essential - these areas will be discussed in this review. Ruthenium polypyridyl complexes, which are able to photooxidise DNA and act as luminescent DNA probes, may serve as a suitable photophysical payload for a TFO system and the developments in this area in the context of DNA triplexes will also be reviewed.

Fig. 1. (a) A-DNA, (b) B-DNA.

Fig. 2. Canonical DNA structure and non-canonical structures including (A) duplex, (B) triplex, (C) G-quadruplex and i-motif and (D) hairpin. Reprinted from H. Tateishi-Karimata and N. Sugimoto, Chem. Commun., 2020, 56, 2379.

Fig. 3. Schematic drawing of a triplex forming oligonucleotide that specifically recognises a DNA sequence, with the TFO binding in the major groove of the DNA duplex. “Reprinted from Coord. Chem. Rev., 257, Tarita Biver, Stabilisation of non-canonical structures of nucleic acids by metal ions and small molecules, 2765–2783, Copyright (2013), with permission from Elsevier.”

Fig. 4. Intermolecular triplexes and canonical base triplets. (a) Polypyrimidine triplexes Y–R:Y (b) polypurine triplexes R–R:Y Reprinted from K. M. Vasquez and P. M. Glazer Triplex-forming oligonucleotides: principles and applications, Q. Rev. Biophys., 35, 89–107, copyright 2002, with permission from Cambridge University Press.

Fig. 5. 3D Representation of and schematic diagram of (a) triplex A (intramolecular antiparallel, PDB ID 134D), (b) triplex B (intramolecular parallel, PDB ID 149D) (c) triplex C (intermolecular parallel, PDB ID 1BWG). The TFO is displayed in red and the DNA duplex is in green. In the schematic diagrams, Watson–Crick hydrogen bonding is displayed using lines with Hoogsteen bonds illustrated in dashed lines.

Fig. 6. Schematic representation of (a) triplexes A (PDB ID 134D) and (b) triplex B (PDB ID 149D). The arrows indicate the four thymine that are reported in the analysis, but do not bind to any complementary base.

Fig. 7. (a) 3D and (b) schematic representations of the G–T:A triplet of the triplex B. Green indicate the duplex bases, guanine and adenine, while the orange base is the guanine of the TFO.

Fig. 8. Representation of sugar rings of B-DNA (circle) and A-DNA (crosses) based on pseudorotation and torsion angle. Reproduced with permission from R. E. Dickerson, International Tables for X-ray Crystallography, Volume F: Macromolecular Crystallography, ed. M. G. Rossmann, E. Arnold (International Union of Crystallography, Chester, U.K. (2001).

Fig. 9. Base modifications in parallel triplexes. (a) 5-Methyl-cytosine, (b) 2′-O-methyl-pseudoisocytidine, (c) 6-oxo-cytosine, (d) 5-methyl-6-oxo-cytosine, (e) α-AP, (f) β-AP, (g) 2′-aminoethoxy-thymine, (h) N⁴-3-acetamidopropyl-cytosine, (i) N⁴-6-aminopyridinyl-cytosine, (j) 5-propynyl-cytosine, (k) 5-propynyl-uracil, (l) 5-bromo-cytosine, (m) 5-iodo-cytosine, (n) 5-bromo-uridine, (o) 2′-O-methyl-2-thio-uridine, (p) 2-thio-thymidine, (q) 6-amino-5-nitropyridin-2-one, (r) N7-glycosilated-guanine, (s) P1-guanine, (t) inosine.

Fig. 10. Base modification for anti-parallel triplexes. (a) 7-deaza-xanthine, (b) 6-thioguanine, (c) 9-deaza-guanine, (d) 7-deaza-guanine, (e) 7-chloro-7deaza-guanine, (f) 8-aza-7-deaza-guanine, (g) PhdG, (h) 8-oxo-adenine, (i) N⁶-methyl-8-oxo-adenine, (j) AY-d(Y-NH₂), (k) AY-d(Y-Cl).

Fig. 11. Phosphate backbone modifications. (a) Phosphorothioates, (b) DEED, (c) DMAP, (d) guanidino, (e) methylthiourea, (f) methyl-phosphonates, (g) PNHME, (h) azido-phosphoramidate, (i) tosyl sulfonyl phosphoramidite, (j) PNA.

Fig. 12. Sugar backbone modifications. (a) LNA, (b) ENA, (c) 2′-OMe, (d) 2′-AE.

Fig. 13. (Left) DNA triplex groove binders and (right) DNA triplex intercalators. Adapted with permission from D. P. Arya, Acc. Chem. Res., 2011, 44, 134–146. Copyright 2011 American Chemical Society.

Fig. 14. Example of structure of a TINA intercalating unit. Reprinted with permission from I. Géci, V. V. Filichev and E. B. Pedersen, Bioconjug. Chem., 2006, 17, 950–957. Copyright 2006 American Chemical Society.

Fig. 15. (A) Optical sensor based on hairpin triplex structure (4) of a target gene (6) by the reconfiguration of a fluorophore/quencher-modified triplex DNA hairpin structure and the release of the stem forming oligonucleotide (5). (B) A triplex DNA hairpin moiety (X) containing an aptamer sequence used as an optical aptasensor that binds the target (7) with subsequent formation of a hairpin excited structure (8). Reprinted and adapted from Triplex DNA Nanostructures: From Basic Properties to Applications Y. Hu, A. Cecconello, A. Idili, F. Ricci, and I. Willner, pages 15210–15233, Copyright (2017), Angew. Chem.

Fig. 16. Chemical structures of the various SI-PPCs.

Fig. 17. Important ruthenium complexes and binding modes Reproduced from Cardin C. J., Kelly J. M. & Quinn S. J. Photochemically active DNA-intercalating ruthenium and related complexes-insights by combining crystallography and transient spectroscopy. Chem. Sci.8, 4705–4723 (2017).

Fig. 18. Jablonski diagram indicating the electronic transition from the excited to the ground state, depending on the solvent. Reproduced from Di Pietro M. L., La Ganga G., La Nastasi F. & Puntoriero F. Ru(ii)-dppz derivatives and their interactions with DNA: thirty years and counting. Appl. Sci.11, (2021).

Maria Dalla Pozza

Ahmad Abdullrahman

Christine J. Cardin

Gilles Gasser

James P. Hall