Folding and misfolding pathways of G-quadruplex DNA

G-quadruplexes adopt various folding topologies, but information on their folding pathways remains scarce. Here, we used electrospray mass spectrometry to detect and quantify the specifically bound potassium ions, and circular dichroism to characterize the stacking topology of each ensemble. For human telomeric (hTel) sequences containing the d((GGGTTA)₃GGG) core, K⁺ binding affinity and cooperativity strongly depends on the chosen construct. The shortest sequences bind only one K⁺ at low KCl concentration, and this 2-quartet G-quadruplex is antiparallel. Flanking bases increase the K⁺ binding cooperativity. To decipher the folding pathways, we investigated the kinetics of K⁺ binding to telomeric (hybrid) and c-myc (parallel) G-quadruplexes. G-quadruplexes fold via branched pathways with multiple parallel reactions. Up to six states (one ensemble without K⁺, two ensembles with 1-K⁺ and three ensembles with 2-K⁺) are separated based on their formation rates and ion mobility spectrometry. All G-quadruplexes first form long-lived misfolded structures (off-pathway compared to the most stable structures) containing one K⁺ and two quartets in an antiparallel stacking arrangement. The results highlight the particular ruggedness of G-quadruplex nucleic acid folding landscapes. Misfolded structures can play important roles for designing artificial G-quadruplex based structures, and for conformational selection by ligands or proteins in a biological context.