G4 are guanine-rich non-canonical nucleic acids conformations in eukaryotic genomes. The cellular, genomic and physiological significance of G4s was skeptical a decade ago. Computational and experimental methods have offered new perspectives into their roles. Biophysical research has demonstrated relationship between the G4 sequence content, topology, and thermodynamic stability. G4s are primarily located in genomic areas such as telomeres, enhancers, and gene promoters. G4s are involved in transcriptional regulation and chromatin mediated epigenetic genome changes. Abnormal formation, stabilization or resolution of G4 structures can contribute to several human diseases development. G4 sequences have been researched as target for therapy in several diseases. Several G4 targets have passed clinical approval. Thus G4 sequences have research potential both in basic biological and clinical research. The current review aims to update the current literature on the biophysical, cellular and disease roles of G4 sequences. Further, we discuss in detail its roles in cancer and therapy. This update will provide valuable insights into their structural and biology insights thus facilitating clinical applications.