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G-triplexes are non-canonical DNA structures formed by G-rich sequences with three G-tracts. Putative G-triplex-forming sequences are expected to be more prevalent than putative G-quadruplex-forming sequences. However, the research on G-triplexes is rare. In this work, the effects of molecular crowding and several physiologically important metal ions on the formation and stability of G-triplexes were examined using a combination of circular dichroism, thermodynamics, optical tweezers and calorimetry techniques. We determined that molecular crowding conditions and cations, such as Na + , K + , Mg 2+  and Ca 2+ , promote the formation of G-triplexes and stabilize these structures. Of these four metal cations, Ca 2+  has the strongest stabilizing effect, followed by K + , Mg 2+ , and Na +  in a decreasing order. The binding of K +  to G-triplexes is accompanied by exothermic heats, and the binding of Ca 2+  with G-triplexes is characterized by endothermic heats. G-triplexes formed from two G-triad layers are not stable at physiological temperatures; however, G-triplexes formed from three G-triads exhibit melting temperatures higher than 37°C, especially under the molecular crowding conditions and in the presence of K +  or Ca 2+ . These observations imply that stable G-triplexes may be formed under physiological conditions.

Divalent cations and molecular crowding buffers stabilize G-triplex at physiologically relevant temperatures