GENERATION OF A NUCLEASE TARGETING PARALLEL G-QUADRUPLEX FOR SPECIFIC CUTTING OF DOUBLE STRANDED DNA

Abstract

G-quadruplex is a secondary structure of DNA or RNA that plays an important role in many biological processes such as replication, transcription, translation and elongation of telomeres. Therefore, the G-quadruplex structure has emerged as a target molecule for drugs designation in biomedical applications. In this study, a novel nuclease which was developed can specifically target parallel G-quadruplex and play its catalytic function at a specific position. The novel nuclease was genetically generated by fusing a parallel G-quadruplex-recognized RHAU53 peptide motif with a catalytic protein domain of Fok1, resulting in the generation of RHAU53-Fok1. The fusion protein could be expressed in E. coli under IPTG inducer and purified by the his-tag affinity chromatography. Interestingly, RHAU53-Fok1 can selectively bind a parallel G-quadruplex and cut a double stranded DNA next to it. Cleavage of double stranded DNA by RHAU53-Fok1 showed multiple cutting sites on the substrate, resulting in a major fragment and several minor fragments. The novel nuclease provides a useful tool for recognizing and mapping the G-quadruplex structure in the genome.