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The landscape of nucleic acid modification technologies is rapidly evolving, with chemoselective postsynthetic labeling strategies emerging as indispensable chemical tools for generating functionalized oligonucleotides (ONs). These methods greatly rely on reactions between groups such as amine/thiol or clickable handles like azide/alkyne with cognate reaction partners. However, achieving precise covalent labeling in the presence of competing functionalities and preserving ON integrity, particularly in RNA, presents significant challenges. Here, we present an innovative chemoenzymatic platform for DNA and RNA labeling that leverages the unique chemoselective reactivity of terminal glycine-modified (Gly-tag) nucleotide analogs with o -substituted benzaldehyde substrates equipped with strategic hydrogen bond acceptors. The Gly-tagged nucleotide analogs (d G TTP and G UTP) serve as excellent substrates for DNA and RNA polymerases and terminal uridylyl transferase, thereby allowing the incorporation of the reactive Gly-tag at desired positions into DNA and RNA ONs. Notably, o -substituted benzaldehyde substrates, bearing a proximal oxyacetamide moiety, facilitate efficient postenzymatic conjugation, enabling site-selective installation of functionalities including affinity tags, fluorescent probes and clickable groups with good yields and remarkable selectivity. Taken together, this chemoenzymatic methodology represents a new toolkit for late-stage ON labeling, opening up new avenues for advancing nucleic acid applications in diagnostics and biotechnology.
Pal et al. (Wed,) studied this question.