This article provides a detailed procedure for the chemical synthesis and characterization of photoswitchable hydrazone phosphoramidite and its incorporation into oligodeoxynucleotides. The synthesis starts with commercially available deoxyuridine, followed by conversion of the 4-oxo into a 4-chloro moiety via Appel reaction to install the key hydrazone group in the absence of base. The hydrazone phosphoramidite building block is compatible with the conventional amidite chemistry protocols for solid-phase synthesis of oligodeoxynucleotides. Our method expands the current nucleotide pool by adding a novel, functional DNA building block that is suitable for a broad spectrum of applications, including the regulation of DNA-enzyme interactions and DNA synthesis by irradiation with cell-friendly blue light. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of photoswitchable hydrazone phosphoramidite Basic Protocol 2: Synthesis and purification of oligodeoxynucleotides containing the hydrazone photoswitch Basic Protocol 3: Primer extension assay for functionality studies of hydrazone cytidine.
Keywords: hydrazone modification; nucleic acids; phosphoramidite; photoswitch; solid phase synthesis.
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