In contrast to the moderate thermodynamic stability of G4 structures \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$(\Delta{G_U} < 3\,{k_B}T)$\end{document} the folding energy barriers are relatively large \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$(\Delta G_F^\ddagger \sim 15\,{k_B}T)$\end{document}, resulting into a slow folding process (several hours were necessary to reach thermodynamic equilibrium in our experimental conditions). Such high folding energy cost can be explained by an extreme flexibility and large degree of freedom of G4-forming sequences. Indeed, the formation of a planar G-quartet, necessary building block for G4s, may require large structural arrangements of the unfolded single-stranded DNA chain to bring together four guanine bases separated by as many as 20 nucleotides. Interestingly, we find similar folding barriers for all G4 conformations populating the energy landscape. This observation may indicate that the transition state separating the unfolded and folded states is likely to be very similar for all G4 conformations observed here. Although our experiments do not provide any direct structural features of the G4 transition state, we, however, may envision it as an electrostatically stabilized compact structure with similar global fold as for the folded G4s, but lacking most of the native contacts.