Imaging multiple DNA repair enzymes in living cells based on framework nucleic acid fluorescence nanoprobe

Abstract

DNA repair enzymes play pivotal roles in DNA damage repair. Imaging multiple DNA repair enzymes in living cells is significant for DNA damage repair-related biological study and disease diagnosis. Here, we constructed a framework nucleic acids (FNAs) fluorescent nanoprobe for imaging multiple DNA repair enzymes in living cells. Uracil DNA glycosylase (UDG) and human apurinic/apyrimidinic endonuclease 1 (APE1) were used as the target analytes. Two double-stranded DNA probes (dsDNA1 and dsDNA2) were designed. dsDNA1 contained four uracil (U) bases for UDG recognition and was labeled with black hole quencher2 (BHQ2) /cyanine5 (Cy5). dsDNA2 contained one apurinic/apyrimidinic (AP) site for APE1 recognition and was labeled with black hole quencher1 (BHQ1) /fluorophore carboxyfluorescein (FAM). To obtain the FNAs fluorescent nanoprobe, dsDNA1 and dsDNA2 were attached to two vertices of a DNA tetrahedron (a type of FNAs). Under the action of UDG and APE1, dsDNA1 and dsDNA2 were both dissociated, generating fluorescence of Cy5 and FAM. Among them, Cy5 indicated the activity of UDG and FAM indicated the activity of APE1. This method had good sensitivity to UDG and APE1, and the detection limits were 0.0012 U/mL and 0.057 U/mL, respectively. This method also exhibited high selectivity for UDG and APE1. When the FNAs fluorescent nanoprobe was endocytosed, it had little toxicity to normal and cancer cells. Furthermore, this method successfully achieved multiple imaging UDG and APE1 in cancer cells. This study provides a novel strategy for imaging multiple DNA repair enzymes and holds great potential in biological applications and clinical diagnosis.