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Pymol image of a triplex DNA structure (PDB: 1BWG). The arrows are going from the 5' end to the 3' end.

Triple-stranded DNA is a DNA structure in which three oligonucleotides wind around each other and form a triple helix. In triple stranded-DNA, one strand binds to a B-form DNA double helix through Hoogsteen base pair or reversed Hoogsteen hydrogen bonds.

Structure

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Most stable triple-base pairing in triple stranded DNA. Rx-Ry: Watson and Crick base pair binding. Ry-Rz: Hoogesteen base pair binding.

Hoogesteen Base Pairing

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A thymine (T) nucleobase can bind to a Watson–Crick base-pairing of T-A by a Hoogsteen hydrogen bond. The thymine attaches to the adosine (A) of the origin double stranded DNA to create a T-A*T base-triplet.[1] In acidic conditions, a protonated cytosine, represented as C+, can form a base-triplet with a C-G pair through Hoogsteen base-pairing, forming C-G*C+. The TA*T and CG*C+ base pairs are the most stabilizing triplet-base pairing that can form, while a TA*G and CG*G are the most destabilizing triplet-base pairing.[2]

Intermolecular and Intramolecular Formation

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Function

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Triplex Forming Oligonucleotides (TFO)

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TFOs are triplex forming molecules that bind to the major groove of the double stranded DNA to form intramolecular triplex DNA structures. They attempt to modulate gene activity in vivo. In peptide nucleic acid (PNA), the sugar-phosphate backbone is replaced with a protein-like backbone. PNAs form P-loops while interacting with duplex DNA, forming triplex with one DNA strand displacing the other. Very unusual recombination or parallel triplexes, or R-DNA, have been assumed to form under RecA protein in the course of homologous recombination.[3]

TFOs bind specifically to homopurine-homopyrimidine regions that are often common in promoter and intron sequences of genes influencing cell signaling.[4] TFOs can inhibit transcription by binding with high specificity to the DNA helix, thereby blocking the binding and function of transcription factors for particular sequences. By using highly specific DNA segments to target TFO regions, expression of genes can be controlled.[5] This application has novel implications in site-specific mutagenesis and gene therapy. The observed inhibition of transcription can also have negative health effects like its role in the recessive, autosomal gene for Friedreich’s Ataxia.[6] In Fredrick’s Ataxia, triplex DNA formation impairs the expression of intron 1 of the FXN gene. This results in the degeneration of the nervous system and spinal cord, impairing the movement of the limbs.[7] To combat this triplex instability, nucleotide excision repair proteins (NERs) have been shown to recognize and repair triple-stranded DNA structures, reinstating full availability of the previously inhibited and unstable gene.[8]

TFOs are promising gene-drugs that can be employed in an anti-gene strategy. In human prostate cancer cells, a transcription factor Ets2 is over-expressed and thought to drive forward the growth and survival of cells in such excess. Carbone et al. designed a sequence-specific TFO to the Ets2 promoter sequence that down-regulated the gene expression and led to a slowing of cell growth and cell death.[9] Changxian et al. have also presented a TFO targeting the promoter sequence of bcl-2, a gene inhibiting apoptosis.[10]

Genetic Instability

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Considerable research has been funneled into the biological implications relating to the presence of triplex DNA, more specifically H-DNA, in the major breakpoint regions (Mbr) and double-strand-breakpoints (DBS) of certain genes. For example, major points of breakage in the P1 promoter of the c-MYC gene have been found neighboring polypurine mirror-repeat H-DNA forming sequences among other non-B DNA structure forming sequences. Cases of genetic instability were also observed in the F1 offspring of transgenic mice after incorporation of human H-DNA-forming sequences paired with Z-DNA sequences into their genomes where no instability was previously reported.[11] Additionally, formation of R.R.Y. triplex conformations have been observed at the Mbr of the bcl-2 gene which is associated with follicular lymphomas.[11][12] Long tracts of GAA·TTC have also been observed to form very stable triplex structures. Interactions between these two triplex structures, termed sticky DNA, has been shown to interrupt transcription of the X25, or frataxin gene. As decreased levels of the protein frataxin is associated with Friedreich's ataxia, formation of this instability has been suggested to be the basis for this genetic disease. .[13][14]

  1. ^ Rhee, Sangkee; Han, Zong-jin; Liu, Keliang; Miles, H. Todd; Davies, David R. (1999). "Structure of a Triple Helical DNA with a Triplex−Duplex Junction‡". Biochemistry. 38 (51): 16810–16815. doi:10.1021/bi991811m. ISSN 0006-2960.
  2. ^ Mergny, Jean Louis; Sun, Jian Sheng; Rougee, Michel; Montenay-Garestier, Therese; Barcelo, Francisca; Chomilier, Jacques; Helene, Claude (1991-10-08). "Sequence specificity in triple helix formation: experimental and theoretical studies of the effect of mismatches on triplex stability". Biochemistry. 30 (40): 9791–9798. doi:10.1021/bi00104a031. ISSN 0006-2960.
  3. ^ Frank-Kamenetskii, M. D.; Mirkin, S. M. (1995-01-01). "Triplex DNA structures". Annual Review of Biochemistry. 64: 65–95. doi:10.1146/annurev.bi.64.070195.000433. ISSN 0066-4154. PMID 7574496.
  4. ^ Brazdova, Marie (December 1, 2016). "p53 Specifically Binds Triplex DNA In Vitro and in Cells". PLoS One. 11: e0167439.
  5. ^ Faria, M.; Wood, C.D; Perrouault, L.; Nelson, J.S; Winter, A.; White, M.R.H.; Helene, C.; Giovannangeli, C. (2000). "Targeted inhibition of transcription elongation in cells mediated by triplex-forming oligonucleotides". PNAS. 97: 3862–3867. Bibcode:2000PNAS...97.3862F. doi:10.1073/pnas.97.8.3862. PMC 18107.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  6. ^ Sakamoto N.; Chastain, P.D; Parniewski, P.; Ohshima, K.; Pandolfo, M.; Griffith, J.D; Wells, R.D. (1999). "Sticky DNA: Self-Association Properties of Long GAA·TTC Repeats in R·R·Y Triplex Structures from Friedreich's Ataxia". Molecular Cell. 3 (4): 465–475. doi:10.1016/s1097-2765(00)80474-8. PMID 10230399.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  7. ^ Bacolla, A.; Wells, R.D. (2009). "Non-B DNA Conformations as Determinants of Mutagenesis and Human Disease". Human Carcinogenisis. 48: 273–285. doi:10.1002/mc.20507. PMID 19306308.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  8. ^ Kaushik Tiwari, Meetu; Adaku, Nneoma; Peart, Natoya; Rogers, Faye A. (2016-06-13). "Triplex structures induce DNA double strand breaks via replication fork collapse in NER deficient cells". Nucleic Acids Research. 44 (16): 7742–7754. doi:10.1093/nar/gkw515. ISSN 0305-1048. PMC 5027492. PMID 27298253. {{cite journal}}: no-break space character in |last= at position 8 (help)CS1 maint: PMC format (link)
  9. ^ Carbone, G. M. (2004-08-03). "Triplex DNA-mediated downregulation of Ets2 expression results in growth inhibition and apoptosis in human prostate cancer cells". Nucleic Acids Research. 32 (14): 4358–4367. doi:10.1093/nar/gkh744. ISSN 1362-4962. PMC 514370. PMID 15314206.{{cite journal}}: CS1 maint: PMC format (link)
  10. ^ Shen, Changxian; Rattat, Dirk; Buck, Andreas; Mehrke, Gerhard; Polat, Bülent; Ribbert, Hanno; Schirrmeister, Holger; Mahren, Bettina; Matuschek, Christiane (2003-02). "Targeting bcl-2 by Triplex-Forming Oligonucleotide—A Promising Carrier for Gene–Radiotherapy". Cancer Biotherapy and Radiopharmaceuticals. 18 (1): 17–26. doi:10.1089/108497803321269296. ISSN 1084-9785. {{cite journal}}: Check date values in: |date= (help)
  11. ^ a b Wang, Guliang; Vasquez, Karen M. (July 2014). "Impact of Alternative DNA Structures of DNA Damage, DNA Repair, and Genetic Instability" (PDF). National Institute of Health. 19: 143–151. doi:10.1016/j.dnarep.2014.03.017.
  12. ^ Raghavan, Sathees C.; Chastain, Paul; Lee, Jeremy S.; Hegde, Balachandra G.; Houston, Sabrina; Langen, Ralf; Hsieh, Chih-Lin; Haworth, Ian S.; Lieber, Michael R. (March 2005). "Evidence for a Triplex DNA Conformation at the bcl-2 Major Breakpoint Region of the t(14;18) Translocation" (PDF). Journal of Biological Chemistry. 280: 22749–22760.
  13. ^ Wang, Guliang; Vasquez, Karen M. (July 2004). "Naturally occurring H-DNA-Forming sequences are mutagenic in mammalian cells" (PDF). PNAS. 101: 13448–13453. doi:10.1073/pnas.0405116101.
  14. ^ Vetcher, Alexandre A.; Napierala, Marek; Iyer, Ravi R.; Chastain, Paul D.; Griffith, Jack D.; Wells, Robert D. (July 2002). "Sticky DNA, a Long GAA-GAA-TTC Triplex That Is Formed Intramolecularly, in the Sequence of Intron 1 of the Frataxin Gene" (PDF). Journal of Biological Chemistry. 277: 39217–39227. doi:10.1074/jbc.M205209200.{{cite journal}}: CS1 maint: unflagged free DOI (link)