These structures are not forgotten due to lack of interest but rather because they are difficult to study. Until now, there has been only one published structure of an antiparallel triplex, and that was 30 years ago.
Triple helices of DNA (triplexes) form when a third strand is bound in the major groove of a double helix (duplex). There is evidence suggesting that triplex exist in the genome and play a role in the regulation of gene expression. In fact, the addition of an exogenous DNA strand to form a triplex in a regulatory region of a gene has been used to inhibit its expression, making these structures of clear pharmacological interest.
There are two types of triplexes based on the orientation of the third strand with respect to the duplex: parallel and antiparallel. The formation of the parallel triplex, which has been more extensively studied, requires acidic pH. In contrast, the antiparallel triplex requires sequences rich in purines (guanines and adenines), which tend to aggregate and form quadruplex structures (four-stranded helices). To prevent quadruplex formation and form more stable triplexes, TINA (Triplex Intercalating Nucleic Acids) was designed. TINA is a molecule that binds between two nucleotides of the third strand of a triplex and intercalates between two consecutive triads.
In collaboration with Dr. V. Filichev's group at Massey University in New Zealand, we have studied an antiparallel triplex with a TINA group attached to the third strand. The insertion of TINA has two effects: (i) it significantly stabilizes the triplex and (ii) causes a broad signal dispersion in the NMR spectrum. The latter has been crucial for assigning the spectra and, consequently, determining the structure. The structure of antiparallel triplexes has been overlooked for many years, not because it is uninteresting but due to the difficulty in assigning NMR spectra of this structure. Until now, only one antiparallel triplex structure had been determined and it was published 30 years ago.
The structure we have published provides a detailed understanding of how TINA intercalates and the reasons of its stabilizing effect on the triplex. This knowledge will contribute to designing and developing more efficient DNA molecules in triple helix formation.
M. Garavís, P. J. B. Edwards, I. Serrano-Chacón, O. Doluca, V. V. Filichev and C. González*. Understanding Intercalative Modulation of G-Rich Sequence Folding: Solution Structure of a TINA-Conjugated Antiparallel DNA Triplex. Nucleic Acids Res., in press, 2024.
