SWITCH-G4 · Selective Wavelength-Induced Targeted Control of DNA-Homo G4s

Guanine-rich (G-rich) oligonucleotides can fold into G-Quadruplexes (G4s), four-stranded DNA structures involved in the fine regulation of key cellular processes. G4s are found in telomeres, oncogene promoters (kRAS, cMYC, BCL2), and non-coding RNA, where they likely play important roles in transcription and translation. Despite extensive research, the precise regulatory function of G4s remains unclear, and key questions such as whether G4 formation actively drives gene expression or is a byproduct of it are still unresolved. A significant limitation to advancing this understanding is the lack of chemical tools that offer the temporal and spatial control necessary for studying G4 dynamics in living cells. This proposal aims to address this challenge by developing a light-responsive tool that enables precise temporal control of G4 folding in relevant promotor region, in living cells. By using UV/visible light as an external trigger, this tool will allow dynamic studies on the effects of G4 formation on gene expression, offering the possibility of controlling the secondary structure folding on-demand. The core of this approach is the synthesis of light-responsive DNA primers, which will be incorporated into photo-responsive plasmids via NEEL, a novel plasmid resynthesis method. The modifications introduced are designed to dynamically favor or inhibit the formation of stable G4 structures by manipulating the assembly of G-tetradsthe core element required for G4 folding. The tool, SWITCH-G4, will be validated using the cKIT promoter model and then expanded to study other G4-forming promoters, such as kRAS, cMYC, and VEGF. This project will provide unprecedented spatial and temporal resolution in the study of G4s, advancing our understanding of their regulatory roles in cells and potentially offering new therapeutic insights.