Biological Background on Triplex Formation

Nucleic acid triplex formation - biochemistry and binding rules

Triplex-formation requires a double-stranded nucleotide in Watson-Crick configuration (duplex) and a single-stranded nucleotide sequence that is acting as the third strand:


Concept of triplex formation


The third strand binds in the major groove of the duplex forming Hoogsteen and reverse Hoogsteen hydrogen bonds with the purines of the duplex, respectively. This also determines the orientation of the third strand with respect to the purines in the duplex:


Triplex motifs


There are six nucleotide triads that allow the formation of two hydrogen bonds between the purines in the duplex and the nucleotides binding in the major groove. These nucleotide triads are used in the canonical ruleset for triplex formation, i.e.
  • the pyrimidine motif, [TC] - where thymines and cytosines bind parallel
  • the purine motif, [GA] - where guanines and adenines bind anti-parallel
  • the purine-pyrimidine motif, [GT] - where guanines and thymines bind either parallel or anti-parallel with respect to the purines in the duplex


Triplex triads

For additional information we refer to our review on nucleic acid triplex formation published in RNA biology (Landes Bioscience - open access article).