Development of a Pseudocellular System to Quantify Specific Interactions Determining the G-Quadruplex Function in Cells

Intracellular chemical microenvironments, including ion concentrations and molecular crowding, play pivotal roles in cell behaviors, such as proliferation, differentiation, and cell death via regulation of gene expression. However, there is no method for quantitative analysis of intracellular environments due to their complexity. Here, we have developed a system for highlighting the environment inside of the cell (SHELL). SHELL is a pseudocellular system, wherein small molecules are removed from the cell and a crowded intracellular environment is maintained. SHELL offers two prominent advantages: (1) It allows for precise quantitative biochemical analysis of a specific factor, and (2) it enables the study of any cell, thereby facilitating the study of target molecule effects in various cellular environments. Here, we used SHELL to study G-quadruplex formation, an event that implicated cancer. We show that G-quadruplexes are more stable in SHELL compared with in vitro conditions. Although malignant transformation perturbs cellular K+ concentrations, environments in SHELL act as buffers against G-quadruplex destabilization at lower K+ concentrations. Notably, the buffering effect was most pronounced in SHELL derived from nonaggressive cancer cells. Stable G-quadruplexes form due to the binding of the G-quadruplex with K+ in different cancer cells. Furthermore, the observed pattern of G-quadruplex-induced transcriptional inhibition in SHELL is consistent with that in living cells at different cancer stages. Our results indicate that ion binding to G-quadruplexes regulates gene expression during pathogenesis.