G-quadruplex is a secondary structure of nucleic acids that plays crucial roles in many significant biological processes. Potential G-quadruplex-forming sequences exist widely in various regions of the genome such as telomeres and gene promoters. In spite of the fact that G-quadruplex can be readily assembled from a single-stranded segment of DNA, its formation from duplex DNA is very difficult under physiological conditions because Watson-Crick interactions in guanine rich segments need to be weakened first. It is demonstrated in our studies that intrastrand G-quadruplex generated from a perfectly matched guanine-rich duplex in a circular DNA as a result of significant quadruplex stabilization and duplex destabilization created by the combined actions of negative DNA supercoiling and molecular crowding conditions.