Here we report efficient and selective post-synthesis conjugation strategies, based on 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 molecules such as fluorophores.  The acquired fluorophore-labeled nucleic acids, prepared from the phosphoramidation reactions, demonstrated conjugation 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 post-synthesis 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 non-specific hybridization in nucleic acids. Intrinsic hybridization specificity of nucleic acids was, however, conserved in the two-step phosphoramidation reaction. The reaction of site-specific conjugating nucleic acids at the 5'-end was supported by fluorescence quenching and UV melting studies of fluorophore-labeled DNA. Finally, phosphoramidation-prepared nucleic acid conjugates were further modified by chemical reactions such as Michael addition and azide-alkyne cycloaddition to synthesize peptide-oligonucleotide conjugates successfully trafficked into human A549 cells. The two-step phosphoramidation-based, effective and site-specific conjugation method has the potential to expedite critical research including delivery, visualization, quantification, structural determination, localization and distribution of nucleic acids in vivo and in vitro.

Effective and site-specific phosphoramidation reaction for universal synthesis of nucleic acid conjugates

248th National Meeting (2014)

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