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A DNA triplex is formed when pyrimidine or purine bases occupy the major groove of the DNA double Helix forming Hoogsteen pairs with purines of the Watson-Crick basepairs. Intermolecular triplexes are formed between triplex-forming oligonucleotides (TFO) and target sequences on duplex DNA. Intramolecular triplexes are the major elements of H-DNAs, unusual DNA structures that are formed in homopurine-homopyrimidine regions of supercoiled DNAs.

Edited: 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 be a triplex forming oligonucleotide (TFO), but could also be a neighboring chromosome. 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.

TFOs are triplex forming molecules that bind to the major groove of the double stranded DNA to form intramolecular triplex DNA structures. TFOs bind specifically to homopurine-homopyrimidine regions. TFOs can inhibit transcription by actively competing with the binding of the transcription factor. Because of the high specificity of the triplex forming molecule, TFOs have been of interest in inhibiting transcription of genes. 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.

In addition to impairing or inhibiting transcription, triplexes have higher frequencies of double strand breaks, which can cause genomic rearrangements like insertions, translocations, inversions, and gross deletions. This genomic instability is a result of the triplex DNA supercoiling to overcome its highly unfavorable thermodynamic constraints.