Significance Guanine-rich nucleic acids fold into a four-stranded structure named G-quadruplex that has implications in essential cellular processes, pharmaceutical applications, and nanodevices. We found a unique type of G-quadruplex that contains a G-vacancy and is stabilized by guanine derivatives such as te physiological concentration of GTP by fill-in at the G-vacancy of a guanine base. In response to changes in the concentration of guanine derivatives, this type of G-quadruplex is able to manipulate the tracking activity of protein on DNA as exemplified by a DNA polymerase-catalyzed DNA synthesis. Because guanine derivatives are natural metabolites in cells, such G-quadruplexes may potentially play an environment-responsive regulation in cellular processes, a functional property not found in the canonical G-quadruplexes.