Effects of single and double mutations on the MYC promoter G-quadruplex using a custom G4 DNA microarray:...

Parallel DNA G-quadruplexes (G4) are key regulators of oncogene transcription. The MYC promoter G-quadruplex (MycG4) is a prototype parallel G4 and promising anticancer drug target. However, effects of DNA damage or mutations on their conformational landscape remain elusive. Systematic analysis of mutational effects is challenging because permutating G4-forming sequences creates a vast sequence space inaccessible to most experimental methods. Herein, using a custom G4-DNA microarray, we systematically examine all 2 145 possible single and double mutations of the MYC promoter G4. The results show no single or even double mutation completely prevents MYC G-quadruplex formation, emphasizing its exceptional robustness. Mutated MycG4 sequences can form G-quadruplexes with vacancies or bulges without the need to replace the damaged G-runs. Both length and position of a G-run determine its resilience against mutations. Intriguingly, our results reveal an efficient compensation mechanism for mutations involving nearby redundant G-residues to preserve G4 formation. Moreover, the most disruptive mutations involve two nonadjacent G-runs, which cannot be repaired by a single G-tract but can still be compensated by redundant guanines. These results provide critical insights into G-quadruplex folding, structural resilience, and damage tolerance, with implications for gene regulation and G4-targeted drug design.