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Triple-stranded DNA (also known as H-DNA or Triplex-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 (Watson–Crick base-pairing) through Hoogsteen base pair or reversed Hoogsteen hydrogen bonds.

Hoogesteen Base Pairing
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. 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.

Intermolecular and Intramolecular Formations
There are two classes of triplex DNA: intermolecular and intramolecular formations. Intermolecular triplex refers to triplex formation between a duplex and a different strand of DNA. The third strand can either be from a neighboring chromosome or a triplex forming oligonucleotide (TFO). Intramolecular triplex DNA is formed from a duplex with homopurine and homopyrimidine strands with mirror repeat symmetry. The level of supercoiling in DNA influences the level of intramolecular triplex formation. There are two different types of intramolecular triplex DNA: H-DNA and H*-DNA. Formation of H-DNA is stabilized under acidic conditions and in the presence of divalent cations such as Mg2+. In this conformation, the homopyrimidine strand in the duplex bends back to bind to the purine strand in a parallel fashion. The base triads used to stabilize this conformation are T-A-*T and C-G*C+. The cytosine in this base triad needs to be protonated in order to form this intramolecular triple helix, which is why this conformation is stabilized under acidic conditions. H*-DNA has favorable formation conditions at neutral pH and in the presence of divalent cations. This intramolecular conformation is formed from the binding of the homopurine and purine strand of the duplex in an antiparallel fashion. It is stabilized by T-A*A and C-G*G base triplets.

Function
Triple-stranded DNA is implicated in the regulation of several genes. Once, the c-myc gene was extensively mutated to examine the role that triplex DNA structure, versus linear sequence, plays in gene regulation. A c-myc promoter element, termed the nuclease-sensitive element or NSE, can form tandem intramolecular triplexes of the H-DNA type and has a repeating sequence motif (ACCCTCCCC)4. The NSE when mutated was examined for transcriptional activity and for intra- and intermolecular triplex forming ability. The transcriptional activity of mutant NSEs can be predicted by the element's ability to form H-DNA and not by repeat number, position, or the number of mutant base pairs.

Triplex Forming Oligonucleotides (TFO)
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.

TFOs bind specifically to homopurine-homopyrimidine regions that are often common in promoter and intron sequences of genes influencing cell signaling (Brazdova). 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. 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. 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. 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 (Tiwari).

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. Changxian et al. have also presented a TFO targeting the promotor sequence of bcl-2, a gene inhibiting apoptosis.

Genetic Instability
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, in addition to other non-B DNA sequences found neighboring the P1 promoter of the c-MYC gene, polypurine mirror-repeat H-DNA forming sequences were found and are associated with the major breakpoint hotspots of this region. 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. Additionally, formation of R.R.Y. triplex conformations have been observed at the Mbr of the bcl-2 gene. Formation of these structures has been posited to cause the t(14;18) translocation observed in many cancers and most follicular lymphomas. This observation has led to research that indicated a substantial decrease in translocation events can be observed after blocking the formation of H-DNA by altering the sequence of this region slightly. 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.

History
Triple-stranded DNA was a common hypothesis in the 1950s when scientists were struggling to discover DNA's true structural form. Watson and Crick (who later won the Nobel Prize for their double-helix model) originally considered a triple-helix model, as did Pauling and Corey, who published a proposal for their triple-helix model in 1953, as well as fellow scientist Fraser. However, Watson and Crick soon identified several problems with these models:


 * Negatively charged phosphates near the axis repel each other, leaving the question of how the three-chain structure stays together.
 * In a triple-helix model (specifically Pauling and Corey's model), some of the van der Waals distances appear to be too small.

Fraser's model differed from Pauling and Corey's in that in his model the phosphates are on the outside and the bases are on the inside, linked together by hydrogen bonds. However, Watson and Crick found Fraser's model to be too ill-defined to comment specifically on its inadequacies.

Triple-stranded DNA was also described in 1957, when it was thought to occur in only one in vivo biological process: as an intermediate product during the action of the E. coli recombination enzyme RecA. Its role in that process is not understood.

Using nucleic acid segments that bind to the DNA duplexes to form triple strands as a way of regulating gene expression is under separate investigation by biotechnology companies and Yale University.