Two-quartet G-quadruplexes formed by DNA sequences containing four contiguous GG runs

The DNA sequence containing four contiguous GG runs (G₂N_xG₂N_yG₂N_zG₂, G₂ sequence) has the potential to form a two-quartet G-quadruplex. However, the prevalence, structure, and function of G₂ sequences have not been well-studied. Here, bioinformatics analysis reveals the abundance of G₂ sequences in the human genome and their enrichment in promoter regions. The density of G₂ sequences in the genome and promoters is much higher than that of the G₃ sequence (G₃N_xG₃N_yG₃N_zG₃). Experiments show that the conformations and thermal stabilities of the two-quartet G-quadruplexes of G₂ sequences are highly sensitive to the length and composition of the loops. Among the two-quartet G-quadruplexes, the parallel G-quadruplex with a loop length of 1 and the antiparallel G-quadruplex with a loop length of 3 show high thermal stabilities. Additionally, the stable parallel G-quadruplexes are stacked into intermolecular higher-order structures. This work determines the prevalence of G₂ sequences in the human genome and demonstrates that the G-quadruplex structures for certain loop lengths and compositions may be stable in vivo. Thus, more attention should be paid to the structure and function of the two-quartet G-quadruplex.