title: Quantification of total phosphorothioate in bacterial DNA by a bromoimane-based fluorescent method.
abstract: The discovery of phosphorothioate (PT) modifications in bacterial DNA has challenged our understanding of conserved phosphodiester backbone structure of cellular DNA. This exclusive DNA modification in bacteria is not found in animal cells yet, and its biological function in bacteria is still poorly understood. Quantitative information about the bacterial PT modifications is thus important for the investigation of their possible biological functions. In this study, we have developed a simple fluorescence method for selective quantification of total PTs in bacterial DNA, based on fluorescent labeling of PTs and subsequent release of the labeled fluorophores for absolute quantification. The method was highly selective to PTs and not interfered by the presence of reactive small molecules or proteins. The quantification of PTs in an E. coli DNA sample was successfully achieved using our method and gave a result of about 455 PTs per million DNA nucleotides, while almost no detectable PTs were found in a mammalian calf thymus DNA. With this new method, the content of phosphorothioate in bacterial DNA could be successfully quantified, serving as a simple method suitable for routine use in biological phosphorothioate related studies.
authors.affiliation: Department of Chemistry, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Tsinghua University, Beijing, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Xiao
authors.affiliation: Department of Chemistry, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Tsinghua University, Beijing, China. xiang-yu@tsinghua.edu.cn.
authors.forename: Yu
authors.initials: Y
authors.lastname: Xiang
mesh_terms: D000818:Animals; D002417:Cattle; D004247:DNA / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D005456:Fluorescent Dyes / Q000737:chemistry*; D054735:Phosphorothioate Oligonucleotides / Q000032:analysis* / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D054735:Phosphorothioate Oligonucleotides; D004247:DNA; C427203:calf thymus DNA
keywords: Bacterial DNA; Bromobimane; Fluorescence measurement; Phosphorothioate