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

Abstract

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+, Mg2+ and Ca2+, promote the formation of G-triplexes and stabilize these structures. Of these four metal cations, Ca2+ has the strongest stabilizing effect, followed by K+, Mg2+, and Na+ in a decreasing order. The binding of K+ to G-triplexes is accompanied by exothermic heats, and the binding of Ca2+ 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 Ca2+. These observations imply that stable G-triplexes may be formed under physiological conditions.

TGF‐β‐induced fibrotic stress increases G‐quadruplex formation in human fibroblasts

“…Physiologically relevant cations such as K + and Na + play a central role in G‐quadruplex stabilization, where divalent cations such as Ca 2+ have been reported to stabilize intermediates in the folding of G4s . The observed increase in G‐quadruplex DNA structures in TGF‐β‐stimulated scar‐forming Dupuytren fibroblasts as opposed to unstimulated controls could be due to this altered ion homeostasis .…”

Combining a Ru(II) “Building Block” and Rapid Screening Approach to Identify DNA Structure-Selective “Light Switch” Compounds

“…G-rich triplexes have been shown to form under physiological conditions, allowing for an intermediate 3 dimensional structure that lacks the fourth G track to form a G-quadruplex. 52 The G-quadruplexes have the same G-rich repeating sequence but differ by two bases at both the 5′ and 3′ ends of the sequence. Substituting the two 5′ and two 3′ adenosines for thymidines inverts the folding direction of the G-quadruplex.…”

Discovery of “folded DNA” structures in human cells: Potential drug targets

“…These may constitute independent folds, or they might instead be intermediates that are formed during the folding and unfolding of G‐quadruplexes . The formation of G‐triplex structures is promoted by Na + , K + , Ca 2+ and Mg 2+ (the effect of bivalent cations is specific for triplexes, and they do not promote formation of G‐quadruplexes) …”

“…14 The formation of G-triplex structures is promoted by Na + , K + , Ca 2+ and Mg 2+ (the effect of bivalent cations is specific for triplexes, and they do not promote formation of G-quadruplexes). 15 Biffi et al 3 generated an "engineered, structure-specific" antibody to quantitatively visualize DNA G-quadruplex structures in human cells. The cells showed punctate staining in their nuclei that was not present without antibody, and disappeared after treatment with DNase, but not RNase.…”