title: Sensitive detection of intracellular sumoylation via SNAP tag-mediated translation and RNA polymerase-based amplification.
abstract: The covalent attachment of small ubiquitin-like modifier (SUMO) to target proteins, defined as sumoylation, is an important post-translational modification that regulates diverse cellular processes and many human diseases. However, functional analysis of sumo modification is usually hampered by the lack of sensitive methods for measuring extremely low abundance of specific sumoylated target in the cells. Here, we develop an ultrasensitive method for intracellular sumoylation assay based on SNAP tag (a mutant of O6-alkylguanine-DNA alkyltransferase)-mediated translation and RNA polymerase-based amplification. Intracellular sumo modification is first converted to the double-stranded DNA (dsDNA) containing the specific T7 promoter sequence via the covalent conjugation of SNAP tag with its substrate benzyl guanidine derivate; then, the dsDNA is extensively transcribed by T7 RNA polymerase to produce large amounts of RNAs, which are easily monitored using the RNA intercalating dye RiboGreen and a standard fluorometer. This method exhibits excellent specificity and high sensitivity and can detect as little as 5 pg of sumoylated p53 proteins, which has improved by as much as 1000-fold than that in the conventional Western blotting assay. Moreover, this method can measure intracellular sumoylation under different physiological conditions. Due to the common translation and amplification module, this method can be further extended to detect a variety of sumoylated proteins and other ubiquitin-like modifications in the cells and might provide a powerful tool for comprehensive analysis of the functions of sumoylation and other ubiquitin-like modifications in the fundamental biological processes and many human diseases.
authors.affiliation: Single-molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Yang
authors.forename: Chun-yang
authors.initials: CY
authors.lastname: Zhang
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry; D006147:Guanine / Q000031:analogs & derivatives; D007364:Intercalating Agents / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques; D011401:Promoter Regions, Genetic; D012313:RNA / Q000378:metabolism; D058207:Sumoylation; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics
chemical_list: D005456:Fluorescent Dyes; D007364:Intercalating Agents; D016159:Tumor Suppressor Protein p53; D014764:Viral Proteins; D006147:Guanine; D012313:RNA; D004247:DNA; D019883:Alkyl and Aryl Transferases; C050956:DNA alkyltransferase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases