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

Hong-Xin Jiang,Ting Zhao,Hai-Wei Fu,De-Ming Kong,Jibin Abraham Punnoose,Yunxi Cui,Hanbin Mao,Deepak Koirala

doi:10.1038/srep09255

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.

Highlights

G-triplexes are non-canonical DNA structures formed by G-rich sequences with three G-tracts
As G-triplet and G-quartet share similar stacking and loop geometry, it is reasonable to assume that circular dichroism (CD) signals reflecting the strand orientation in Sequencea
We used CD spectroscopy to examine the secondary structures of these six oligonucleotides in the presence of different metal ions, as well as under dilute and molecular crowding conditions

Summary

Introduction

G-triplexes are non-canonical DNA structures formed by G-rich sequences with three G-tracts. A G-rich sequence with only three G-tracts may form a G-triplex with stacked G-triads, in which a central guanine connects with two other guanines by Hoogsteen-like hydrogen-bonds (Figure 1)[7,8,9,10]. The formation of these G-triplexes has been hypothesized[11,12,13], and experiments have suggested that they might be intermediates during folding or unfolding of G-quadruplexes[9,13,14,15,16,17]. Given that intracellular environment is molecularly crowded, in which approximately 30,40% of the cellular volume can be occupied by macromolecules, here we wish to determine whether crowded buffers will promote the formation of G-triplexes and stabilize these structures, similar to those reported for G-quadruplex structures[18,19,20,21,22,23]

Methods

Results

Conclusion