title: Effective and site-specific phosphoramidation reaction for universally labeling nucleic acids.
abstract: Here we report efficient and selective postsynthesis labeling strategies, based on an advanced phosphoramidation reaction, for nucleic acids of either synthetic or enzyme-catalyzed origin. The reactions provided phosphorimidazolide intermediates of DNA or RNA which, whether reacted in one pot (one-step) or purified (two-step), were directly or indirectly phosphoramidated with label molecules. The acquired fluorophore-labeled nucleic acids, prepared from the phosphoramidation reactions, demonstrated labeling efficacy by their F/N ratio values (number of fluorophores per molecule of nucleic acid) of 0.02-1.2 which are comparable or better than conventional postsynthesis fluorescent labeling methods for DNA and RNA. Yet, PCR and UV melting studies of the one-step phosphoramidation-prepared FITC-labeled DNA indicated that the reaction might facilitate nonspecific hybridization in nucleic acids. Intrinsic hybridization specificity of nucleic acids was, however, conserved in the two-step phosphoramidation reaction. The reaction of site-specific labeling nucleic acids at the 5'-end was supported by fluorescence quenching and UV melting studies of fluorophore-labeled DNA. The two-step phosphoramidation-based, effective, and site-specific labeling method has the potential to expedite critical research including visualization, quantification, structural determination, localization, and distribution of nucleic acids in vivo and in vitro.
authors.affiliation: Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 80708, Taiwan.
authors.forename: Yu-Chih
authors.initials: YC
authors.lastname: Su
authors.affiliation: Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 80708, Taiwan.
authors.forename: Hsing-Yin
authors.initials: HY
authors.lastname: Chen
authors.affiliation: Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 80708, Taiwan.
authors.forename: Ni Chien
authors.initials: NC
authors.lastname: Ko
authors.affiliation: Department of Biochemistry, Faculty of Medicine, College of Medicine Kaohsiung Medical University, Kaohsiung 80708, Taiwan.
authors.forename: Chi-Ching
authors.initials: CC
authors.lastname: Hwang
authors.affiliation: Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 80708, Taiwan.
authors.forename: Min Hui
authors.initials: MH
authors.lastname: Wu
authors.affiliation: Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 80708, Taiwan.
authors.forename: Li-Fang
authors.initials: LF
authors.lastname: Wang
authors.affiliation: Department of Biological Science and Technology, National Chiao Tung University, Hsin-Chu 300, Taiwan.
authors.forename: Yun-Ming
authors.initials: YM
authors.lastname: Wang
authors.affiliation: Cardiovascular Center, National Taiwan University Hospital Yun-Lin Branch, Dou-Liu 640, Taiwan.
authors.forename: Sheng-Nan
authors.initials: SN
authors.lastname: Chang
authors.affiliation: Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. Electronic address: enchwa@kmu.edu.tw.
authors.forename: Eng-Chi
authors.initials: EC
authors.lastname: Wang
authors.affiliation: Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. Electronic address: tzupinw@cc.kmu.edu.tw.
authors.forename: Tzu-Pin
authors.initials: TP
authors.lastname: Wang
mesh_terms: D000577:Amides / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D010766:Phosphorylation; D012313:RNA / Q000737:chemistry*
chemical_list: D000577:Amides; D005456:Fluorescent Dyes; D012313:RNA; D004247:DNA; D016650:Fluorescein-5-isothiocyanate
keywords: Labeling; Nucleic acid; Phosphoramidation; Postsynthesis; Site specific