Abstract
G-quadruplexes are nucleic acids structures stabilized by physiological concentration of potassium ions. Because low stability G-quadruplexes are hardly detectable by mass spectrometry, we optimized solvent conditions: isopropanol in a triethylamine/hexafluoroisopropanol mixture highly increased G-quadruplex sensitivity with no modification of the physiological G-quadruplex conformation. G-quadruplexes/G-quadruplex-ligand complexes were also correctly detected at concentration as low as 40 nM. Detection of the physiological conformation of G4s and their complexes opens up the possibility to perform high-throughput screening of G-quadruplex ligands for the development of drug molecules effective against critical human diseases.
Highlights
G -quadruplexes (G4s) are noncanonical nucleic acid structures, which form in guanine (G)-rich sequences by G-quartet stacking, and are stabilized by potassium (K+), the most relevant intracellular monovalent cation.[1,2] G4s may adopt different conformations, i.e., parallel, antiparallel, or hybrid, depending on their strand orientation.[3]
Different approaches have been developed: (i) substitution of K+ with the volatile NH4+ ion, which fits in the G4;17,18 G4s folded in NH4+ often adopt nonphysiological conformations.[19] (ii) G4 folding in physiological concentration of K+ and removal of the noncoordinated ions by filtration or ethanol precipitation;[20,21] this approach is suitable only for stable G4s with slow unfolding kinetics in low K+ concentration. (iii) G4 folding in MScompatible amounts of K+ (
TMAA at 120 mM pH 7.4 roughly consists of equimolar amounts of trimethylamine and acetic acid; in contrast, the amount of triethylamine necessary to neutralize a solution of 120 mM-HFIP is considerably lower (
Summary
AIn bold is the intensity of the prevalent adduct in the corresponding sample. The numbers represent the intensity of the free DNA (no K) or K+ adducts (1K+, 2K+, 3K+, 4K+). The base peak, i.e., the peak with the highest intensity within each spectrum, is shown
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