Direct detection of 8-oxoG G-quadruplex using Surveyor nuclease and GQ specific ligands

Abstract

G-quadruplex (GQ) is one of the secondary structures of DNA in the guanine-rich sequences. GQ regulates DNA replication, transcription, and enzyme activity in the body. Research shows when DNA sequence that makes GQ has damage, it can cause problems with the biological function of GQ, and lead to cancer and other diseases. In particular, the guanine in the DNA sequence that makes GQ is weak to oxidative damage, making a mutation called 8-Oxoguanine (8-oxoG). Consequently, 8-oxoG GQ becomes unstable and induces some diseases such as cancer or hypoxia. In these reasons, we have developed a platform for detecting 8-oxoG mutations in G-quadruplex using enzymes and GQ-specific binding ligands. We first confirmed that there was a difference between the structural stability of wild-type GQ and 8-oxoG GQ using smFRET. Screening was conducted to find enzymes that could cleave 8oxoG GQ using various enzymes. Enzymes such as CEL1 endonuclease, hOGG1, APE1, Exo3 nuclease and S1 nuclease did not cleave 8oxoG of GQ and only the Surveyor nuclease cleaved that. We performed gel analysis according to various conformations of wild type and 8-oxoG type. As a result, only 8-oxoG GQ structure and single-strand DNA were cut off. Based on these results, GQ-specific binding ligands, Crystal violet and N-Methyl mesoporphyrin IX were treated to detect GQ not cleaved by the enzyme. Enzyme cleaves 8oxoG GQ and the ligands detect the remaining wild type GQ. In this research, we could find 8-oxoG GQ specific enzyme and quantify amount and ratio of 8-oxoG GQ using difference of GQ structural stability. It can be an accurate and easy mutation detection platform.