Effective and site-specific phosphoramidation reaction for universally labeling nucleic acids

Yu Chih Su, Hsing Yin Chen, Ni Chien Ko, Chi Ching Hwang, Min Hui Wu, Li Fang Wang, Yun-Ming Wang, Sheng Nan Chang, Eng Chi Wang*, Tzu Pin Wang

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

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.

Original languageEnglish
Pages (from-to)118-128
Number of pages11
JournalAnalytical Biochemistry
Volume449
Issue number1
DOIs
StatePublished - 15 Mar 2014

Keywords

  • Labeling
  • Nucleic acid
  • Phosphoramidation
  • Postsynthesis
  • Site specific

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