Molecular crowding promotes the aggregation of parallel structured G-quadruplexes.

G-quadruplexes are widely distributed in cells and are usually essential in mediating biological processes. The intracellular environment is often in a state of molecular crowding, and the current research considerably focuses on the effect of molecular crowding on the conformation of telomeric G-quadruplexes. However, G-quadruplex-forming oligonucleotides are primarily located in the promoter region of the proto-oncogene and on mRNA inside the cell and are reported to fold into parallel structures. Thus, studying the interaction mechanism between ligands and parallel structured G-quadruplexes under crowding conditions is crucial for the design of drugs targeting G-quadruplexes. In our study, molecular crowding was simulated through polyethylene glycol with an average molecular weight of 200 (PEG200) to investigate the parallel structure of the canonical G-quadruplexes c-KIT1, c-MYC, and 32KRAS and their interactions with ligands. Circular dichroism (CD) spectral scanning, fluorescence resonance energy transfer (FRET), and native polyacrylamide gel electrophoresis (PAGE) analysis revealed that molecular crowding failed to induce oligonucleotides to form parallel G-quadruplex structures in the explored model sequences while induced telomeric G-rich sequences to form antiparallel G-quadruplexes in solution without K. Molecular crowding did not induce changes in their parallel structures but promoted the formation of G-quadruplex aggregates. Moreover, to some extent, molecular crowding also induced a looser structure of the monomer G-quadruplexes. Further studies showed that molecular crowding did not alter the binding stoichiometry of the ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino [4,3,2-kl] acridinium methosulfate (RHPS4) to c-KIT1, while it inhibited its interaction with parallel structured G-quadruplexes. This work provides new insights into developing anticancer drugs targeting parallel structured G-quadruplexes.